Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-PD-Li ANTIBODIES AND FUSION PROTEINS THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
100011 This application claims the benefit of and priority to U.S. Provisional
Patent Application
No. 63/134,412, filed January 6, 2021, the disclosure of which is incorporated
herein by
reference in its entirety for all purposes.
FIELD OF THE INVENTION
100021 The invention relates generally to antibodies, recombinant sialidase
fusion proteins, and
antibody conjugates, and their use in the treatment of cancer.
BACKGROUND
100031 Programmed death-ligand 1 (PD-L1) also known as cluster of
differentiation 274
(CD274) or B7 homolog 1 (B7-H1) is a protein that in humans is encoded by the
CD274 gene.
Upregulation of PD-Li may allow certain cancers to evade the host immune
system. An
analysis of 196 tumor specimens from patients with renal cell carcinoma found
that high tumor
expression of PD-Li was associated with increased tumor aggressiveness and a
4.5-fold
increased risk of death (Thompson et al. (2004) PROC. NATL. ACAD. So. USA
101(49) 17174-
17179). PD-Li expression is detected in many human cancers, including bladder,
breast,
cervical, esophageal, gastric, kidney, lung, ovary and pancreatic cancer (Wang
et al. (2016)
ONCO. TARGETS THER, 9:5023-5039). For certain cancers, expression of PD-Li is
associated
with reduced numbers of tumor infiltrating lymphocytes and poor prognosis
(Ohaegbulam et al.
(2015) TRENDS MOL. MED. 21(1): 24-33). A number of anti-PD-L1 antibodies have
already
been approved in the United States for treating a variety of cancers. For
example, atezolizumab
has been approved for use in, for example, urothelial carcinomas, non-small
cell lung cancers
(NSCLC), triple-negative breast cancers, and small cell lung cancers,
durvalumab has been
approved for use in, for example, urothelial carcinomas, and NSCLCs, and
avelumab has been
approved for use in Merkel cell carcinomas, urothelial carcinomas, and renal
cell carcinomas.
Other PD-Li antibodies are in still in development as immuno-oncology
therapies and are
showing good results in clinical trials including for treating NSCLC and
melanoma (Akinleye et
al. (2019) J. HEMATOL. ONCOL, 12(1):92).
100041 A growing body of evidence supports roles for glycans, and sialoglycans
in particular, at
various pathophysiological steps of tumor progression. Glycans regulate tumor
proliferation,
invasion, hematogenous metastasis and angiogenesis (Fuster et al. (2005) NAT.
REV. CANCER
5(7): 526-42). The sialylation of cell surface glycoconjugates is frequently
altered in cancers,
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resulting in the expression of sialylated tumor-associated carbohydrate
antigens. The expression
of sialylated glycans by tumor cells is often associated with increased
aggressiveness and
metastatic potential of a tumor (Julien S., Delannoy P. (2015) "Sialic Acid
and Cancer", In:
Taniguchi N., Endo T., Hart G., Seeberger P., Wong CH. (eds) Glycoscience:
Biology and
Medicine. Springer, Tokyo. https://doi.org/10.1007/978-4-431-54841-6 193).
[0005] It has recently become apparent that Siglees (sialic acid-binding
immunoglobulin-like
lectins), a family of sialic acid binding lectins, play a role in cancer
immune suppression by
binding to hypersialylated cancer cells and mediating the suppression of
signals from activating
NK cell receptors, thereby inhibiting NK cell-mediated killing of tumor cells
(Jandus etal.
(2014) J. CLIN. INVEST. 124: 1810-1820; Laubli et al (2014) PROC. NATL. ACAD.
So. USA 111:
14211-14216; Hudak et al. (2014) NAT. CHEM. BIOL. 10: 69-75). Likewise,
enzymatic removal
of sialic acids by treatment with sialidase can enhance NK cell-mediated
killing of tumor cells
(Jandus, supra; Hudak, supra; Xi ao et al. (2016) PROC. NATL. ACAD. Sci. USA
113(37): 10304-
9).
[0006] Cancer immunotherapy with immune checkpoint inhibitors, including
antibodies that
block the PD-1/PD-L1 pathway, has improved the outcome of many cancer
patients. However,
despite advances that have been made to date, many patients do not respond to
currently
available immune checkpoint inhibitors. Accordingly, there is still a need for
effective
interventions that overcome the immune suppressive tumor microenvironment and
for treating
cancers associated with hypersialylated cancer cells.
SUMMARY OF THE IN VEN T1ON
[0007] The invention is based, in part, upon the discovery of anti-PD-L1
antibodies that impact
or otherwise down regulate signaling mediated by PD-1 or PD-Li. In the
appropriate
circumstances, the antibodies can remove the PD-1 or PD-Li-mediated repression
of a subject's
immune system to mediate the removal of non-natural cells, for example,
cancerous cells.
[0008] The invention is also based, in part, upon the discovery that it is
possible to produce
fusion proteins containing a sialidase enzyme and an anti-PD-Li immunoglobulin
or a portion
thereof, e.g., an antigen-binding domain and/or an immunoglobulin Fc domain,
and/or antibody
conjugates including a sialidase enzyme and an anti-PD-Li antibody or a
portion thereof, e.g , an
antigen-binding domain and/or an immunoglobulin Fc domain. The sialidase
enzyme portion of
the fusion protein and/or antibody conjugate may comprise at least one
mutation relative to a
wild-type sialidase. The mutations, or combination of mutations, can improve
the expression,
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activity or both the expression and activity of the sialidase to improve its
use in cancer diagnosis
and/or treatment.
100091 The fusion proteins and/or antibody conjugates have suitable substrate
specificities and
activities to be useful in removing sialic acid and/or sialic acid containing
molecules from the
surface of cancer cells, e.g., PD-Li-expressing cancer cells, and/or removing
sialic acid and/or
sialic acid containing molecules from the tumor microenvironment, and/or
reducing the
concentration of sialic acid and/or sialic acid containing molecules in the
tumor
microenvironment.
100101 Accordingly, in one aspect, the invention provides an isolated antibody
that binds human
PD-Li.
100111 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO. 161,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 162, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 163 (PAL769-VH, h769-VH); and/or an
immunoglobulin light chain variable region comprising a CDRL1 comprising the
amino acid
sequence of SEQ ID NO: 165, a CDRL2 comprising the amino acid sequence of SEQ
ID NO:
142, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 166 (PAL769-
VL,
h769-IF3-VL, h769-tm2-VL, h769-tm3-VL).
100121 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 250,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 251, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 163 (PAL769-VH); and/or an
immunoglobulin light
chain variable region comprising a CDRL1 comprising the amino acid sequence of
SEQ ID NO:
253, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 254, and a CDRL3
comprising the amino acid sequence of SEQ ID NO: 166 (PAL769-VL).
100131 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 250,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 252, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 163 (h769-VH); and/or an immunoglobulin
light chain
variable region comprising a CDRL1 comprising the amino acid sequence of SEQ
ID NO: 255,
a CDRL2 comprising the amino acid sequence of SEQ ID NO: 254, and a CDRL3
comprising
the amino acid sequence of SEQ ID NO: 166 (h769-IF3-VL, h769-tm2-VL, h769-tm3-
VL).
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100141 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 161, a
CDRH2 comprising the amino acid sequence of SEQ ID NO: 162, and a CDRH3
comprising the
amino acid sequence of SEQ ID NO: 163 (PAL769-VH, h769-VH); and/or an
immunoglobulin
light chain variable region comprising a CDRL1 comprising the amino acid
sequence of SEQ ID
NO: 165, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 142, and a
CDRL3
comprising the amino acid sequence of SEQ ID NO: 203 (h769.T-VL).
100151 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 250,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 252, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 163 (h769-VH); and/or an immunoglobulin
light chain
variable region comprising a CDRL1 comprising the amino acid sequence of SEQ
ID NO: 255,
a CDRL2 comprising the amino acid sequence of SEQ ID NO: 254, and a CDRL3
comprising
the amino acid sequence of SEQ ID NO: 203 (h769.T-VL).
100161 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 129,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 131 (PAL752-VH); and/or an
immunoglobulin light
chain variable region comprising a CDRL1 comprising the amino acid sequence of
SEQ ID NO:
133, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 134, and a CDRL3
comprising the amino acid sequence of SEQ ID NO: 135 (PA1L752-VL).
100171 In certain embodiments, the antibody comprises an immunoglobulin
heavy chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 137,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 138, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 139 (PAL759-VH); and/or an
immunoglobulin light
chain variable region comprising a CDRL1 comprising the amino acid sequence of
SEQ ID NO:
141, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 142, and a CDRL3
comprising the amino acid sequence of SEQ ID NO: 143 (PAL759-VL).
100181 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 145,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 147 (PAL760-VH); and/or an
immunoglobulin light
chain variable region comprising a CDRL1 comprising the amino acid sequence of
SEQ ID NO:
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149, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 150, and a CDRL3
comprising the amino acid sequence of SEQ ID NO: 151 (PAL760-VL).
100191 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 153,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 154, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 155 (PAL767-VH); and/or an
immunoglobulin light
chain variable region comprising a CDRL1 comprising the amino acid sequence of
SEQ ID NO:
157, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 158, and a CDRL3
comprising the amino acid sequence of SEQ ID NO: 159 (PAL767-VL).
100201 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 161,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 168, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 169 (PAL771-VH); and/or an
immunoglobulin light
chain variable region comprising a CDRL1 comprising the amino acid sequence of
SEQ ID NO:
171, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 172, and a CDRL3
comprising the amino acid sequence of SEQ ID NO: 173 (PAL771-VL).
100211 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 175,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 176, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO. 177 (PAL785-VH), and/or an
immunoglobulin light
chain variable region comprising a CDRL1 comprising the amino acid sequence of
SEQ ID NO:
179, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 180, and a CDRL3
comprising the amino acid sequence of SEQ ID NO: 181 (PAL785-VL).
100221 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 183,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 184, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 185 (PAL787-VH); and/or an
immunoglobulin light
chain variable region comprising a CDRL1 comprising the amino acid sequence of
SEQ ID NO:
187, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 188, and a CDRL3
comprising the amino acid sequence of SEQ ID NO: 189 (PAL787-VL).
100231 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 191,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 192, and a CDRH3
comprising
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the amino acid sequence of SEQ ID NO: 193 (PAL788-VH); and/or an
immunoglobulin light
chain variable region comprising a CDRL1 comprising the amino acid sequence of
SEQ ID NO:
195, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 196, and a CDRL3
comprising the amino acid sequence of SEQ ID NO: 197 (PAL788-VL).
100241 In certain embodiments of any of the foregoing antibodies, the CDRs are
interposed
between human or humanized immunoglobulin framework regions.
100251 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising the amino acid sequence of SEQ ID NO: 164 (PAL769-
VH), and an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
167 (PAL769-VL).
100261 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising the amino acid sequence of SEQ ID NO. 199 (h769-
VH), and/or an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
200 (h769-IF3-VL).
100271 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising the amino acid sequence of SEQ ID NO: 199 (h769-
VH), and/or an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
201 (h769-tm2-VL).
100281 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising the amino acid sequence of SEQ ID NO: 199 (h769-
VH), and/or an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
202 (h769-tm3-VL).
100291 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising the amino acid sequence of SEQ ID NO: 199 (h769-
VH), and/or an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
204 (h769.T-VL).
100301 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising the amino acid sequence of SEQ ID NO: 132 (P4L752-
VH), and/or
an immunoglobulin light chain variable region comprising the amino acid
sequence of SEQ ID
NO: 136 (PAL752-VL).
100311 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising the amino acid sequence of SEQ ID NO: 140 (PAL759-
VH), and/or
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an immunoglobulin light chain variable region comprising the amino acid
sequence of SEQ ID
NO: 144 (PAL759-VL).
100321 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising the amino acid sequence of SEQ ID NO: 148 (PAL760-
VH), and/or
an immunoglobulin light chain variable region comprising the amino acid
sequence of SEQ ID
NO: 152 (PAL760-VL).
100331 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising the amino acid sequence of SEQ ID NO: 156 (PAL767-
VH), and/or
an immunoglobulin light chain variable region comprising the amino acid
sequence of SEQ ID
NO: 160 (PAL767-VL).
100341 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising the amino acid sequence of SEQ ID NO. 170 (PAL771-
VH), and/or
an immunoglobulin light chain variable region comprising the amino acid
sequence of SEQ ID
NO: 174 (PAL771-VL).
100351 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising the amino acid sequence of SEQ ID NO: 178 (PAL785-
VH), and/or
an immunoglobulin light chain variable region comprising the amino acid
sequence of SEQ ID
NO: 182 (PAL785-VL).
100361 In certain embodiments, the antibody comprises an immunoglobulin
heavy chain
variable region comprising the amino acid sequence of SEQ ID NO: 186 (P4L787-
VH), and/or
an immunoglobulin light chain variable region comprising the amino acid
sequence of SEQ ID
NO. 190 (PAL787-VL).
100371 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
variable region comprising the amino acid sequence of SEQ ID NO: 194 (PAL788-
VH), and/or
an immunoglobulin light chain variable region comprising the amino acid
sequence of SEQ ID
NO: 198 (PAL788-VL).
100381 In certain embodiments of any of the foregoing antibodies, the antibody
further
comprises a heavy chain constant region (e.g., an IgGl, IgG2, IgG3, and IgG4
heavy chain
constant region) and/or light chain constant region.
100391 In certain embodiments of any of the foregoing antibodies, the antibody
binds to human
PD-Li with a KD of 5 nM or lower, 3 nM or lower, 2.5 nM or lower, 2 nM or
lower, 1 nM or
lower, 0.75 nM or lower, 0.5 nM or lower, 0.1 nM, 0.075 nM, or 0.05 nM or
lower, as measured
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by surface plasmon resonance or bio-layer interferometry. In certain
embodiments, the antibody
also binds to Macaca fascicularis (cynomolgus) PD-Li.
100401 In another aspect, the invention provides an isolated antibody that
competes with any of
the foregoing antibodies for binding to human PD-Li and/or binds to the same
epitope on
human PD-Li as any of the foregoing antibodies.
100411 In another aspect, the invention provides an isolated nucleic acid
comprising a nucleotide
sequence encoding an immunoglobulin heavy chain variable region of any of the
foregoing
antibodies and/or an immunoglobulin light chain variable region of any of the
foregoing
antibodies.
100421 In another aspect, the invention provides an expression vector
comprising: (i) a nucleic
acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain
variable region
of any of the foregoing antibodies; and/or (ii) a nucleic acid comprising a
nucleotide sequence
encoding an immunoglobulin light chain variable region of any of the foregoing
antibodies.
100431 In another aspect, the invention provides a host cell comprising any of
the foregoing
nucleic acids or expression vectors.
100441 In another aspect, the invention provides a fusion protein comprising
(or consisting
essentially of): (a) sialidase enzyme; and (b) an anti-PD-Li immunoglobulin
antigen-binding
domain derived from any of the foregoing antibodies.
100451 In certain embodiments, the sialidase is a human sialidase, e.g., a
recombinant mutant
human sialidase. In certain embodiments, the sialidase comprises: (a) a
substitution or deletion
of a methionine residue at a position corresponding to position 1 of wild-type
human Neu2
(M1); (b) a substitution of a valine residue at a position corresponding to
position 6 of wild-type
human Neu2 (V6); (c) a substitution of a lysine residue at a position
corresponding to position 9
of wild-type human Neu2 (K9); (d) a substitution of an alanine residue at a
position
corresponding to position 42 of wild-type human Neu2 (A42); (e) a substitution
of a proline
residue at a position corresponding to position 62 of wild-type human Neu2
(P62); (f) a
substitution of an alanine residue at a position corresponding to position 93
of wild-type human
Neu2 (A93); (g) a substitution of a glutamine residue at a position
corresponding to position 126
of wild-type human Neu2 (Q126); (h) a substitution of an isoleucine residue at
a position
corresponding to position 187 of wild-type human Neu2 (I187); (i) a
substitution of an alanine
residue at a position corresponding to position 242 of wild-type human Neu2
(A242); (j) a
substitution of a glutamine residue at a position corresponding to position
270 of wild-type
human Neu2 (Q270); (k) a substitution of a serine residue at a position
corresponding to position
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301 of wild-type human Neu2 (S301); (1) a substitution of a tryptophan residue
at a position
corresponding to position 302 of wild-type human Neu2 (W302); (m) a
substitution of a cysteine
residue at a position corresponding to position 332 of wild-type human Neu2
(C332); (n) a
substitution of a valine residue at a position corresponding to position 363
of wild-type human
Neu2 (V363); or (o) a substitution of a leucine residue at a position
corresponding to position
365 of wild-type human Neu2 (L365); or a combination of any of the foregoing
substitutions
100461 In certain embodiments, in the sialidase. (a) the methionine residue at
a position
corresponding to position 1 of wild-type human Neu2 is deleted (AM1), is
substituted by alanine
(M1A), or is substituted by aspartic acid (M1D); (b) the valine residue at a
position
corresponding to position 6 of wild-type human Neu2 is substituted by tyrosine
(V6Y); (c) the
lysine residue at a position corresponding to position 9 of wild-type human
Neu2 is substituted
by aspartic acid (K9D), (d) the alanine residue at a position corresponding to
position 42 of wild-
type human Neu2 is substituted by arginine (A42R) or aspartic acid (A42D), (e)
the proline
residue at a position corresponding to position 62 of wild-type human Neu2 is
substituted by
asparagine (P62N), aspartic acid (P62D), histidine (P62H), glutamic acid
(P62E), glycine
(P62G), serine (P62S), or threonine (P62T), (f) the alanine residue at a
position corresponding to
position 93 of wild-type human Neu2 is substituted by glutamic acid (A93E) or
lysine (A93K),
(g) the glutamine residue at a position corresponding to position 126 of wild-
type human Neu2 is
substituted by leucine (Q126L), glutamic acid (Q126E), phenylalanine (Q126F),
histidine
(Q126H), isoleucine (Q126I), or tyrosine (Q126Y); (h) the isoleucine residue
at a position
corresponding to position 187 of wild-type human Neu2 is substituted by lysine
(I187K); (i) the
alanine residue at a position corresponding to position 242 of wild-type human
Neu2 is
substituted by cysteine (A242C), phenylalanine (A242F), glycine (A242G),
histidine (A242H),
isoleucine (A242I), lysine (A242K), leucine (A242L), methionine (A242M),
asparagine
(A242N), glutamine (A242Q), arginine (A242R), serine (A242S), valine (A242V),
tryptophan
(A242W), or tyrosine (A242Y); (j) the glutamine residue at a position
corresponding to position
270 of wild-type human Neu2 is substituted by alanine (Q270A), histidine
(Q270H),
phenylalanine (Q270F), proline (Q270P), serine (Q270S), or threonine (Q270T);
(k) the serine
residue at a position corresponding to position 301 of wild-type human Neu2 is
substituted by
alanine (S301A), aspartic acid (S301D), glutamic acid (S301E), phenylalanine
(S301F), histidine
(S301H), lysine (S301K), leucine (S301L), methionine (S301M), asparagine
(S301N), proline
(S301P), glutamine (S301Q), arginine (S301R), threonine (S301T), valine
(S301V), tryptophan
(S301W), or tyrosine (S301Y); (1) the tryptophan residue at a position
corresponding to position
302 of wild-type human Neu2 is substituted by alanine (W302A), aspartic acid
(W302D),
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phenylalanine (W302F), glycine (W302G), histidine (W30211), isoleucine
(W3021), lysine
(W302K), leucine (W302L), methionine (W302M), asparagine (W302N), proline
(W302P),
glutamine (W302Q), arginine (W302R), serine (W302S), threonine (W302T), valine
(W302V),
or tyrosine (W302Y); (m) the cysteine residue at a position corresponding to
position 332 of
wild-type human Neu2 is substituted by alanine (C332A); (n) the valine residue
at a position
corresponding to position 363 of wild-type human Neu2 is substituted by
arginine (V363R); or
(o) the leucine residue at a position corresponding to position 365 of wild-
type human Neu2 is
substituted by glutamine (L365Q), histidine (L365H), isoleucine (L365I),
lysine (L365K) or
serine (L365S); or the sialidase comprises a combination of any of the
foregoing substitutions.
For example, the sialidase may comprise a substitution selected from AM1, M1A,
M1D, V6Y,
K9D, A42R, P62G, P62N, P62S, P62T, A93E, Q126Y, I187K, A242F, A242W, A242Y,
Q270A, Q270T, S301A, S301R, W302K, W302R, C332A, V363R, and L365I, or a
combination
of any of the foregoing substitutions.
100471 In certain embodiments, the sialidase comprises: (a) the M1D, V6Y,
P62G, A93E,
I187K, and C332A substitutions; (b) the M1D, V6Y, K9D, A93E, I187K, C332A,
V363R, and
L365I substitutions; (c) the M1D, V6Y, P62N, I187K, and C332A substitutions;
(d) the M1D,
V6Y, I187K, Q270A, S301R, W302K, and C332A substitutions; (e) the M1D, V6Y,
P62S,
I187K, Q270A, S301R, W302K, and C332A substitutions; (f) the M1D, V6Y, P62T,
I187K,
Q270A, S301R, W302K, and C332A substitutions; (g) the MID, V6Y, P62N, I187K,
Q270A,
S301R, W302K, and C332A substitutions; (h) the M1D, V6Y, P62G, A93E, I187K,
S301A,
W302R, and C332A substitutions; (i) the M1D, V6Y, P62G, A93E, Q126Y, I187K,
Q270T, and
C332A substitutions; (j) the M1D, V6Y, P62G, A93E, Q126Y, I187K, and C332A
substitutions;
(k) the M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, and C332A
substitutions; or
(1) the M1D, V6Y, A42R, P62G, A93E, Q126Y, I187K, A242F, Q270T, and C332A
mutations.
100481 In certain embodiments, the sialidase is selected from Neul, Neu2,
Neu3, and Neu4, e.g.,
the sialidase is Neu2.
100491 In certain embodiments, the sialidase has a different substrate
specificity than the
corresponding wild-type sialidase. For example, in certain embodiments the
sialidase can cleave
a2,3, a2,6, and/or a2,8 linkages. In certain embodiments the sialidase can
cleave a2,3 and a2,8
linkages.
100501 In certain embodiments, the sialidase comprises any one of SEQ ID NOs:
48-62, 94, 97,
100, 126, or 234, or an amino acid sequence that has at least 85%, 90%, 95%,
96%, 97%, 98%,
or 99% sequence identity to any one of SEQ ID NOs: 48-62, 94, 97, 100, 126, or
234.
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100511 In certain embodiments, the sialidase comprises mutation or combination
of mutations
set forth in any one of Tables 1-9.
100521 In certain embodiments, the fusion protein further comprises an
immunoglobulin Fc
domain. In certain embodiments, the immunoglobulin Fc domain is derived from a
human IgGl,
IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, or IgM Fc domain, e.g., the
immunoglobulin Fc
domain is derived from a human IgGl, IgG2, IgG3, or IgG4 Fc domain, e.g., the
immunoglobulin Fc domain is derived from a human IgG1 Fc domain.
100531 In certain embodiments, the anti-PD-Li immunoglobulin antigen-binding
domain is
associated (for example, covalently or non-covalently associated) with a
second anti-PD-Li
immunoglobulin antigen-binding domain to produce an anti-PD-Li antigen-binding
site. For
example, in certain embodiments the anti-PD-Li immunoglobulin antigen-binding
domain is an
immunoglobulin heavy chain fragment that is associated with an immunoglobulin
light chain
fragment to produce an anti-PD-Li antigen-binding site. In other embodiments
the anti-PD-Li
immunoglobulin antigen-binding domain is an immunoglobulin light chain
fragment that is
associated with an immunoglobulin heavy chain fragment to produce an anti-PD-
Li antigen-
binding site.
100541 In certain embodiments, the sialidase and the immunoglobulin Fc domain
and/or the anti-
PD-Li immunoglobulin antigen-binding domain are linked by a peptide bond or an
amino acid
linker.
100551 In certain embodiments, the fusion protein comprises any one of SEQ ID
NOs: 205-207,
211, 213, 214, and 219.
100561 In another aspect, the invention provides an antibody conjugate
comprising any of the
foregoing fusion proteins. In certain embodiments, the antibody conjugate
comprises a single
sialidase. In other embodiments, the antibody conjugate comprises two
sialidases, which can be
the same or different. In certain embodiments the antibody conjugate comprises
two identical
sialidases. In certain embodiments, the antibody conjugate comprises a single
anti-PD-Ll
antigen-binding site. In other embodiments, the antibody conjugate comprises
two anti-PD-Li
antigen-binding sites, which can be the same or different. In certain
embodiments, the antibody
conjugate comprises two identical anti-PD-Li antigen-binding sites.
100571 In certain embodiments, the antibody conjugate has a molecular weight
from about 135
kDa to about 165 kDa, or the antibody conjugate has a molecular weight from
about 215 kDa to
about 245 kDa.
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100581 In certain embodiments, the antibody conjugate comprises: (a) a first
polypeptide
comprising an immunoglobulin light chain; (b) a second polypeptide comprising
an
immunoglobulin heavy chain; and (c) a third polypeptide comprising an
immunoglobulin Fc
domain and a sialidase; wherein the first and second polypeptides are
covalently linked together
and the second and third polypeptides are covalently linked together, and
wherein the first
polypeptide and the second polypeptide together define an anti-PD-Li antigen-
binding site. The
third polypeptide may, for example, comprise the sialidase and the
immunoglobulin Fe domain
in an N- to C-terminal orientation. The first polypeptide may, for example,
comprise SEQ ID
NO: 205, the second polypeptide may, for example, comprise any one of SEQ ID
NOs: 206 or
213, and/or the third polypeptide may, for example, comprise any one of SEQ ID
NOs: 207, 211,
214, or 219.
100591 In certain embodiments, the antibody conjugate comprises: (a) a first
polypeptide
comprising a first immunoglobulin light chain; (b) a second polypeptide
comprising a first
immunoglobulin heavy chain and a first sialidase; (c) a third polypeptide
comprising a second
immunoglobulin heavy chain and a second sialidase; and (d) a fourth
polypeptide comprising a
second immunoglobulin light chain; wherein the first and second polypeptides
are covalently
linked together, the third and fourth polypeptides are covalently linked
together, and the second
and third polypeptides are covalently linked together, and wherein the first
polypeptide and the
second polypeptide together define a first anti-PD-Li antigen-binding site,
and the third
polypeptide and the fourth polypeptide together define a second anti-PD-Li
antigen-binding site.
The second and third polypeptides may, for example, comprise the first and
second
immunoglobulin heavy chain and the first and second sialidase, respectively,
in an N- to C-
terminal orientation.
100601 In certain embodiments, the antibody conjugate comprises: (a) a first
polypeptide
comprising a first sialidase, a first immunoglobulin Fe domain, and a first
single chain variable
fragment (scFv); and (b) a second polypeptide comprising a second sialidase, a
second
immunoglobulin Fe domain, and an optional second single chain variable
fragment (scFv);
wherein the first and second polypeptides are covalently linked together, and
wherein the first
scFv defines a first anti-PD-Li antigen-binding site, and the second scFv,
when present, defines
a second anti-PD-Li antigen-binding site The first polypeptide may, for
example comprise the
first sialidase, the first immunoglobulin Fe domain, and the first scFv in an
N- to C-terminal
orientation. The second polypeptide may, for example, comprise the second
sialidase, the
second immunoglobulin Fe domain, and the optional second scFv in an N- to C-
terminal
orientation.
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[0061] In certain embodiments, the antibody conjugate comprises: (a) a first
polypeptide
comprising an immunoglobulin light chain; (b) a second polypeptide comprising
an
immunoglobulin heavy chain and a single chain variable fragment (scFv); and
(c) a third
polypeptide comprising an immunoglobulin Fc domain and a sialidase, wherein
the first and
second polypeptides are covalently linked together and the second and third
polypeptides are
covalently linked together, and wherein the immunoglobulin light chain and
immunoglobulin
heavy chain together define a first anti-PD-Li antigen-binding site and the
scFv defines a second
anti-PD-Ll antigen-binding site. The second polypeptide may, for example
comprise the
immunoglobulin heavy chain and the scFv in an N- to C-terminal orientation.
The third
polypeptide may, for example, comprise the sialidase and the immunoglobulin Fc
domain in an
N- to C-terminal orientation.
[0062] In another aspect, the invention provides an isolated nucleic acid
comprising a nucleotide
sequence encoding at least a portion of any of the foregoing antibodies, any
of the foregoing
fusion proteins, or at least a portion of any of the foregoing antibody
conjugates. In another
aspect, the invention provides an expression vector comprising any of the
foregoing nucleic
acids. In another aspect, the invention provides a host cell comprising any of
the foregoing
expression vectors.
[0063] In another aspect, the invention provides a pharmaceutical composition
comprising any
of the foregoing antibodies, any of the foregoing fusion proteins, or any of
the foregoing
antibody conjugates.
[0064] In another aspect, the invention provides a method of treating cancer
in a subject in need
thereof The method comprises administering to the subject an effective amount
of any of the
foregoing antibodies, any of the foregoing fusion proteins, any of the
foregoing antibody
conjugates, or any of the foregoing pharmaceutical compositions.
[0065] In certain embodiments, the cancer is selected from non-small cell lung
cancer (NSCLC),
melanoma, bladder, breast, cervical, esophageal, gastric, kidney, lung, ovary,
metastatic Merkel
cell carcinoma (MCC), metastatic urothelial carcinoma (UC), and pancreatic
cancer.
[0066] These and other aspects and features of the invention are described in
the following
detailed description and claims.
DESCRIPTION OF THE DRAWINGS
[0067] The invention can be more completely understood with reference to the
following
drawings.
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100681 FIGURE 1 depicts an SDS-PAGE gel showing recombinant human Neul, Neu2,
Neu3,
and Salmonella typhimurium (St-sialidase) under non-reducing and reducing
conditions.
Monomer and dimer species are indicated.
100691 FIGURE 2 is a bar graph showing the enzymatic activity of recombinant
human Neul,
Neu2, and Neu3.
100701 FIGURE 3 is a line graph showing enzymatic activity as a function of
substrate
concentration for recombinant human Neu2 and Neu3 at the indicated pH.
100711 FIGURES 4A-4I depict schematic representations of certain antibody
conjugate
constructs containing a sialidase enzyme, e.g., a human sialidase enzyme, and
an anti-PD-L1
antigen binding site. For each antibody conjugate construct that contains more
than one (e.g.,
two) sialidase, each sialidase may be the same or different. For each antibody
conjugate
construct that contains more than one (e.g., two) anti-PD-Li antigen binding
site, each anti-PD-
Li antigen binding site may be the same or different. For each antibody
conjugate construct that
contains an Fc domain, it is understood that the Fc domain can be a wild type
Fc domain or can
be an engineered Fc domain For example, the Fc domain may be engineered to
contain either a
"knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, or both, to
promote
heterodimerization, or the Fc domain may be engineered to contain one or more
modifications,
e.g., point mutations, to provide any other modified Fc domain functionality.
100721 FIGURE 5 depicts schematic representations of certain antibody
conjugate constructs
containing a sialidase enzyme, e.g., a human sialidase enzyme, and an antigen
binding site. For
each antibody conjugate construct that contains more than one (e.g., two)
antigen binding site,
each antigen binding site may be the same or different. For each antibody
conjugate construct
that contains an Fe domain, it is understood that the Fc domain can be a wild
type Fc domain or
can be an engineered Fc domain. For example, the Fe domain may be engineered
to contain
either a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, or
both, to promote
heterodimerization, or the Fc domain may be engineered to contain one or more
modifications,
e.g., point mutations, to provide any other modified Fc domain functionality.
100731 FIGURES 6A-6D are schematic representations of exemplary fusion protein
conjugates
referred to as a Raptor antibody sialidase conjugate (FIGURE 6A), a Janus
antibody sialidase
conjugate (FIGURE 6B), a Lobster antibody sialidase conjugate (FIGURE 6C), a
Bunk
antibody sialidase conjugate (FIGURE 6D), and a Lobster-Fab antibody sialidase
conjugate
(FIGURE 6E).
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[0074] FIGURE 7 provides a graph showing the fold induction of a PD-1/PD-L1
linked NFAT
driven luciferase reporter by the indicated hybridoma supernatant comprising
anti-PD-Li
antibodies, showing the ability of the antibodies to functionally block the
interaction between
PD-Li and PD-1. FIGURE 7A, FIGURE 7B, and FIGURE 7C represent different
hybridomas where candidate antibodies with fold inductions greater than 4 are
identified.
[0075] FIGURES 8A, 8B, 8C, and 8D provide graphs showing ForteBio octet
binding kinetics
for human PD-Li binding to purified hybridoma antibodies.
[0076] FIGURES 9A, 9B, 9C, and 9D provide graphs showing ForteBio octet
binding kinetics
for cynomolgus PD-Li binding to purified hybridoma antibodies.
[0077] FIGURE 10 provides a graph showing ELISA results indicative of the
ability of purified
hybridoma antibodies to block human PD-1-Fc binding to human PD-Li. The IC50
(nM) for
each antibody is shown
[0078] FIGURE 11 provides a graph showing ForteBio octet binding kinetics for
human PD-Li
(FIGURE 11A and FIGURE 11B) and cynomolgus PD-Li (FIGURE 11C and FIGURE 11D)
binding to chimeric IgG antibodies.
[0079] FIGURE 12 provides a graph showing ELISA results indicative of the
ability of anti-
hPD-L1 chimeric IgGs to block PD-1/PD-L1 interaction. The IC50 (nM) for each
antibody is
shown.
[0080] FIGURE 13 provides size exclusion chromatography (SEC) profiles of
selected chimeric
IgGs. Monomeric percentage of each chimeric antibody is shown.
[0081] FIGURE 14 is a graph showing binding of chimeric PD-L1 antibodies to
HCC827
(FIGURE 14A) and NCI-H292 (FIGURE 14B) lung epithelial cell lines. Binding was
measured using a FACS analysis with a fluorescein-labeled secondary antibody.
MFI = mean
fluorescence intensity. The apparent Kd (nM) for each antibody is shown
[0082] FIGURE 15 is a graph depicting the level of binding and internalization
of chimeric PD-
L1 antibodies on human Monocyte-derived Dendritic Cells (moDC). Cells were
incubated with
the indicated antibodies at 1 nM, 10 nM and 10 nM antibody in the presence or
absence
(no-stim) of PAM3K.
[0083] FIGURE 16 depicts the ability of chimeric PD-Li antibodies to
functionally block the
interaction between PD-Li and PD-1 using the PD-Ll/PD-1 bioassay. Fold
induction was
calculated by dividing the RLU of induced cells minus background by the RLU of
the no
antibody control minus background. Apparent Kd (nM) for each antibody is
indicated.
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100841 FIGURE 17 depicts the specificity of binding of chimeric PD-Li
antibodies to CHO
cells expressing PD-Li (CHO-PD-L1, staining at 10 nM) vs parental CHO cells
(staining at 100
nM).
100851 FIGURES 18A-D are graphs depicting the enhancement of T cell
proliferation and
cytokine response to allogeneic moDC in the presence of the indicated PD-Li
antibodies
compared to isotype control (001-1) Levels of CD4 T cell proliferation (FIGURE
18A), CD8 T
cell proliferation (FIGURE 18B), TNFct (FIGURE 18C) and IEN-y levels (FIGURE
18D) are
depicted.
100861 FIGURE 19 is a graph depicting the enhancement of cytokine response in
T cells to
allogeneic moDC in the presence of indicated PD-Li antibodies compared to
isotype control
(001-1). Levels of IL-2 (FIGURE 19A), IL-4 (FIGURE 19B), IL-6 (FIGURE 19C) and
IL-10
(FIGURE 19D) are depicted.
100871 FIGURE 20 is a graph depicting the enhancement of degranulation in moDC-
T cell
mixed lymphocyte reactions (MLR) in the presence of the indicated PD-Li
antibodies compared
to isotype control (001-1) Levels of soluble Fas Ligand (FIGURE 20A), Granzyme
A
(FIGURE 20B), perforin (FIGURE 20C) and granulysin (FIGURE 20D) are depicted.
100881 FIGURE 21A depicts an alignment between murine 769VH-wt (SEQ ID NO:
164) and
humanized h769VH-mF0 (SEQ ID NO: 199). FIGURE 21B depicts an alignment between
murine 769Vk-wt (SEQ ID NO: 167) and humanized h769Vk-mF0 (SEQ ID NO: 242),
h769Vk-
T53I (SEQ ID NO: 243), h769Vk-A55F (SEQ ID NO: 244), h769Vk-S67Y (SEQ ID NO:
245),
and h769Vk-Y87F (SEQ ID NO: 246). FIGURE 21C depicts an alignment between
humanized
h769Vk-IY (SEQ ID NO: 247), h769Vk-IF2 (SEQ ID NO: 248), h769Vk-tml (SEQ ID
NO:
249), h769Vk-IF3 (SEQ ID NO: 200), h769Vk-tm2 (SEQ ID NO: 201), and h769Vk-tm3
(SEQ
ID NO: 202). Framework regions are shown in grey. Vkappa back mutations that
were made as
part of the humanization are shown in boxes.
100891 FIGURE 22 depicts SEC profiles of selected 769-hIgG1 humanized
variants.
100901 FIGURE 23 provides graphs showing ForteBio octet binding kinetics of
human PD-Li
(FIGURE 23A) and cynomolgus PD-Li (FIGURE 23B) to selected 769-hIgG1 humanized
variants.
100911 FIGURE 24 provides a graph showing the ability of humanized PD-Li
antibodies to
block PD-1/PD-L1 interaction. The IC50 (nM) for each antibody is shown.
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100921 FIGURE 25 provides a graph showing ForteBio octet binding kinetics of
selected 769-
hIgG1 humanized variants to human PD-Li.
100931 FIGURE 26 depicts SEC profiles of selected 769-hIgG1 humanized
variants.
100941 FIGURE 27 provides graphs depicting the ability of selected 769-hIgG1
humanized
variants to enhance T cell response to allogeneic moDC. Levels of CD4 T cell
proliferation
(FIGURE 27A), Granzyme B (FIGURE 27B), and IFN-y (FIGURE 27C) as well as CD8 T
cell proliferation (FIGURE 2711), Granzyme A (FIGURE 27E) and TNFa levels
(FIGURE
27F) are shown.
100951 FIGURE 28 provides graphs depicting the levels of the indicated
cytokines released in
response to selected 769-hIgG1 humanized variants, suggesting that the 769-
hIgG1 humanized
variants are capable of enhancing T cell response to allogeneic moDC. Perforin
(FIGURE
28A), soluble Fas (FIGURE 28B), IL-6 (FIGURE 28C), Granulysin (FIGURE 2811),
soluble
Fas Ligand (FIGURE 28E) and IL-10 levels (FIGURE 28F) are shown.
100961 FIGURE 29 provides graphs depicting the levels of the indicated
cytokines released
from peripheral blood mononuclear cells (PBMCs) in response to the
cytomegalovirus pp65
peptide mix ("CMV pp65") and in the presence of selected 769-hIgG1 humanized
variants. "No
stim" = unstimulated control (i.e., cells not exposed to CMV pp65). Levels of
IL-2 (FIGURE
29A) and TNFa (FIGURE 29B) in pg/ml are shown.
100971 FIGURE 30 provides graphs depicting the levels of the indicated
cytokines released
from peripheral blood mononuclear cells (PBMCs) in response to the
cytomegalovirus pp65
peptide mix ("CMV pp65") and in the presence of selected 769-hIgG1 humanized
variants. `No
stim" = unstimulated control (i.e., cells not exposed to CMV pp65). Levels of
IL-6 (FIGURE
30A) and IL-17A (FIGURE 30B) in pg/ml are shown.
100981 FIGURE 31 provides graphs depicting the levels of the indicated
cytokines released
from peripheral blood mononuclear cells (PBMCs) in response to the
cytomegalovirus pp65
peptide mix ("CMV pp65") and in the presence of selected 769-hIgG1 humanized
variants. "No
stim" = unstimulated control (i.e., cells not exposed to CMV pp65). Levels of
Granzyme A
(FIGURE 31A) and Granzyme B (FIGURE 31B) in pg/ml are shown
100991 FIGURE 32 provides graphs depicting the levels of the indicated
cytokines released
from peripheral blood mononuclear cells (PBMCs) in response to the
cytomegalovirus pp65
peptide mix ("CMV pp65") and in the presence of selected 769-hIgG1 humanized
variants. "No
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Stim- = unstimulated control (i.e., cells not exposed to CMV pp65). Levels of
Perforin
(FIGURE 32A) and Granulysin (FIGURE 32B) in pg/ml are shown.
101001 FIGURE 33 provides graphs depicting the levels of IFN-7 released from
peripheral
blood mononuclear cells (PBMCs) in response to the cytomegalovirus pp65
peptide mix ("CMV
pp65") and in the presence of selected 769-hIgG1 humanized variants. "No stim"
=
unstimulated control (i.e., cells not exposed to CMV pp65).
101011 FIGURE 34 depicts an epitope binning sandwich assay for the antibodies
indicated.
101021 FIGURE 35 depicts the biochemical characterization of a sialidase-anti-
PD-Li
conjugate. FIGURE 35A provides a photograph of the non-reduced and reduced
PAGE of the
purified molecule. FIGURE 35B shows the SEC profile of a sialidase-anti-PD-L1
conjugate
(ASC1) with a demonstrated purity of 89%.
101031 FIGURE 36 provides graphs showing ForteBio octet binding kinetics of
selected 769-
hIgG1 humanized variants, a PD-Li antibody sialidase conjugate (ASC1), and
atezolizumab to
human PD-Li.
101041 FIGURE 37A and B depicts the biochemical characterization of a second
sialidase-anti-
PD-L1 conjugate (ASC3). FIGURE 37A depicts the SEC profile of the second
sialidase-anti-
PD-L1 conjugate (ASC3). FIGURE 37B is a graph showing the relative
fluorescence units
(RFU), indicative of sialidase activity, over increasing substrate
concentration. Three batches of
the purified second sialidase-anti-PD-L1 conjugate, WG7, WG8, and WG9, were
tested and had
similar activity.
101051 FIGURE 38 depicts the SEC profile of a third sialidase-anti-PD-Li
conjugate with an
inactivate sialidase (ASC4 loss of function or LOF).
101061 FIGURES 39A and B depict the biochemical characterization of a fourth
sialidase-anti-
PD-L1 conjugate FIGURE 39A depicts the SEC profile of the fourth sialidase-
anti-PD-L1
conjugate (ASC5). FIGURE 39B is a graph showing the relative fluorescence
units (RFU),
indicative of sialidase activity of the fourth sialidase-anti-PD-L1 conjugate
(ASC5), over
increasing substrate concentration.
101071 FIGURE 40A depicts the SEC profile of a fifth sialidase-anti-PD-L1
conjugate (ASC2).
FIGURE 40B is a graph showing the relative fluorescence units (RFU),
indicative of the
sialidase activity of the fifth sialidase-anti-PD-L1 conjugate (ASC2), over
increasing substrate
concentration. ASC is also depicted.
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[0108] FIGURE 41 provides graphs showing ForteBio octet binding kinetics of
human PD-Li
(FIGURE 41A) and cynomolgus PD-Li (FIGURE 41B) binding to selected sialidase-
anti-PD-
Li conjugates as compared to h769.T-1A, where the second sialidase PD-Li
heterodimer is
ASC2, the third sialidase PD-Li heterodimer is ASC3, and the fourth sialidase
PD-Li
heterodimer is ASC4 LOF.
[0109] FIGURE 42 provides the results of an experiment in which a PD-Li
antibody sialidase
conjugate or a control molecule was bound to PD-Li-expressing cell lines
(HCC827 and NC-
H292), and then exposed to a secondary antibody conjugated to a fluorescent
moiety. The
graphs show mean fluorescence units (MFI) over increasing concentrations of
sialidase-anti-PD-
Li conjugate or control molecule, depicting binding of the indicated ASC or
antibody to
HCC827 (FIGURE 42A) and NCI-H292 (FIGURE 42B) lung epithelial cell lines.
[0110] FIGURE 43 depicts MFI, which is indicative of de-sialylation, over
increasing
concentrations of sialidase-anti-PD-L1 conjugates on K562 cells (FIGURE 43A)
and HT-29
cells (FIGURE 43B).
[0111] FIGURE 44 depicts the in vivo efficacy of the indicated anti-PD-Li
antibody sialidase
conjugates in a mouse syngeneic subcutaneous tumor model. Mean tumor volumes
over 21 days
for the indicated treatments are indicated in FIGURE 44A. Triangles indicate
dosing.
Individual tumor volumes on day 21 are depicted in FIGURE 44B. One-way ANOVA
(* p <
0.05; ** p <0.005; ns Non-significant).
[0112] FIGURE 45 depicts blocking of the interaction between human PD-Li and
human PD-
1-Fc by ASC5, as measured by ELISA. Two independent preparations of ASC5 (Lot
1 and Lot
2) were tested. Results for h769.T-1A and atezolizumab are also shown. Human
PD-1-Fc only
(no Ab; full PD-Li/PD-1 binding) and buffer only (no antibody and no human PD-
I-Fc; no PD-
Ll/PD-1 binding) were used as controls.
[0113] FIGURE 46 depicts blocking of the PD-Li and PD-1 interaction by ASC5,
as measured
by fold induction of a PD-1/PD-L1 linked NFAT driven luciferase reporter.
Results are shown
for three independent preparations of ASC5 (First Lot, Second Lot, and Third
Lot). Results are
also shown for bivalent anti-PD-Li antibodies h769.T-1A and atezolizumab.
[0114] FIGURE 47 depicts the effect of ASC5 on cytokine release in a DC-T co-
culture
experiment. ASC5 was tested at 700 nM (100 mg/ml), h769.T- IA and atezolizumab
at 70 nM
(10 mg/ml), and isotype control (001-1G) at 100 mg/ml. Each data point
represents a separate
DC-T donor pair. FIGURE 47A depicts the fold change of EL-2 for ASC5, h769.T-
1A,
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atezolizumab, and isotype control. FIGURE 47B, FIGURE 47C, and FIGURE 47D show
similar data for IFN-y, IL-8, and MCP1, respectively.
[0115] FIGURE 48 depicts in vivo efficacy of ASC5 and h769.T-1A, each at the
indicated dose,
in a MC38 mouse syngeneic subcutaneous tumor model. Isotype antibody (001-1G)
and
atezolizumab were used as controls. Mean tumor volumes SEM over 18 days are
depicted in
FIGURE 48A. Triangles indicate drug administration. Individual tumor volumes
on day 18 are
depicted in FIGURE 48B. One-way ANOVA (** p <0.005).
[0116] FIGURE 49 depicts in vivo efficacy of ASC5 and h769.T-1A in a CT26
mouse
syngeneic subcutaneous tumor model. Isotype antibody (001-1G) was used as a
control. Tumor
growth inhibition over 18 days is depicted in FIGURE 49A. Individual tumor
volumes on day
18 are depicted in FIGURE 49B. One-way ANOVA (**** p < 0.05; ns Non-
significant).
[0117] FIGURE 50 depicts in vivo efficacy of ASC5, ASC4 LOF, and h769.T-1A,
each at the
indicated dose, in a CT26 mouse syngeneic subcutaneous tumor model. Isotype
antibody (001-
1G) and atezolizumab were used as controls. Mean tumor volumes SEM over 16
days are
depicted in FIGURE 50A. Individual tumor volumes on day 16 are depicted in
FIGURE 50B.
One-way ANOVA (*** p < 0.05; ns Non-significant).
[0118] FIGURE 51A depicts CDR and framework sequences for heavy chain variable
region
sequences SEQ ID NO: 164 and SEQ ID NO: 199. FIGURE 51B depicts CDR and
framework
sequences for light chain variable region sequences SEQ ID NO: 167, SEQ ID NO:
200, SEQ ID
NO: 201, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 242, SEQ ID NO: 243, SEQ
ID NO:
244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, and SEQ
ID NO:
249. Framework sequences are shown in grey.
DETAILED DESCRIPTION
[0119] The invention is based, in part, upon the discovery of anti-PD-L1
antibodies that impact
or otherwise down regulate signaling mediated by PD-1 or PD-Li.
[0120] Furthermore, the invention is also based, in part, upon the discovery
that it is possible to
produce fusion proteins containing a sialidase enzyme and an anti-PD-Li
immunoglobulin or a
portion thereof, e.g., an antigen-binding domain and/or an immunoglobulin Fc
domain, and/or
antibody conjugates including a sialidase enzyme and an anti-PD-Li antibody or
a portion
thereof, e.g., an antigen-binding domain and/or an immunoglobulin Fc domain.
The sialidase
enzyme portion of the fusion protein and/or antibody conjugate may comprise at
least one
mutation relative to a wild-type sialidase. The mutations, or combination of
mutations, can
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improve the expression, activity or both the expression and activity of the
sialidase to improve
its use in cancer diagnosis and/or treatment. The fusion proteins and/or
antibody conjugates
have suitable substrate specificities and activities to be useful in removing
sialic acid and/or
sialic acid containing molecules from the surface of cancer cells, e.g., PD-Li-
expressing cancer
cells, and/or removing sialic acid and/or sialic acid containing molecules
from the tumor
microenvironment, and/or reducing the concentration of sialic acid and/or
sialic acid containing
molecules in the tumor microenvironment.
101211 The invention further relates to pharmaceutical compositions and
methods of using
antibodies, fusion proteins, and/or antibody conjugates to treat cancer.
I. Anti-PD-Li Antibodies
101221 Among other things, the invention provides antibodies that bind PD-Li
and have the
ability to inhibit PD-Li and/or PD-Li mediated downstream activities and,
therefore, are useful
in treating disorders associated with elevated levels of PD-L1, for example,
cancer, for example,
a cancer that evades a subject's immune system via PD-Li mediated suppression
of a subject's
immune system It is believed that, in certain embodiments, the anti-PD-Li
antibodies described
herein disrupt the interaction between PD-Li and PD-1.
101231 In general, antibodies are multimeric proteins that contain four
polypeptide chains. Two
of the polypeptide chains are called immunoglobulin heavy chains (H chains),
and two of the
polypeptide chains are called immunoglobulin light chains (L chains). The
immunoglobulin
heavy and light chains are connected by an interchain disulfide bond. The
immunoglobulin
heavy chains are connected by interchain disulfide bonds. A light chain
consists of one variable
region (VI) and one constant region (CL). The heavy chain consists of one
variable region (VH)
and at least three constant regions (CHi, CH2 and CH3). The variable regions
determine the
binding specificity of the antibody.
101241 Each variable region contains three hypervariable regions known as
complementarity
determining regions (CDRs) flanked by four relatively conserved regions known
as framework
regions (FRs). The extent of the FRs and CDRs has been defined (Kabat, E.A.,
et al (1991)
SEQUENCES OF PRO ____ IEINS OF IMMUNOLOGICAL IN FEREST, FIFTH EDITION, U.S.
Department of
Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al
(1987) J. MOL.
BIOL. 196:901-917). The three CDRs, referred to as CDR3, CDR2, and CDR3,
contribute to the
antibody binding specificity. Naturally occurring antibodies have been used as
starting material
for engineered antibodies, such as chimeric antibodies and humanized
antibodies.
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101251 As used herein, unless otherwise indicated, the term "antibody- is
understood to mean
an intact antibody (e.g., an intact monoclonal antibody) or a fragment
thereof, such as an
antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of
a monoclonal
antibody) or a Fc fragment of an antibody (e.g., an Fc fragment of a
monoclonal antibody),
including an intact antibody, antigen-binding fragment, or Fc fragment that
has been modified,
engineered, or chemically conjugated. Examples of antigen-binding fragments
include Fab,
Fab', (Fab')2, Fv, single chain antibodies (e.g., scFv), minibodies, and
diabodies. Examples of
antibodies that have been modified or engineered include chimeric antibodies,
humanized
antibodies, and multispecific antibodies (e.g., bispecific antibodies). An
example of a
chemically conjugated antibody is an antibody conjugated to a toxin moiety.
101261 As disclosed herein, antibodies of the invention may comprise: (a) an
immunoglobulin
heavy chain variable region comprising the structure CDRHi-CDRH2-CDRH3 and (b)
an
immunoglobulin light chain variable region comprising the structure CDRL1-
CDRL2-CDRL3,
wherein the heavy chain variable region and the light chain variable region
together define a
single binding site for binding PD-Li.
101271 In certain embodiments, an antibody can comprise: an immunoglobulin
heavy chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 161,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 162, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 163, wherein CDRHi, CDRH2, and CDRH3
sequences
are interposed between immunoglobulin FR sequences (PAL769-VH, h769-VH);
and/or an
immunoglobulin light chain variable region comprising a CDRL1 comprising the
amino acid
sequence of SEQ ID NO: 165, a CDRL2 comprising the amino acid sequence of SEQ
ID NO:
142, and a CDRL3 comprising the amino acid sequence of SEQ Ill NO: 166,
wherein the
CDRLi, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR
sequences
(PAL769-VL, h769-1F3-VL, h769-tm2-VL, h769-tm3-VL).
101281 In certain embodiments, an antibody can comprise: an immunoglobulin
heavy chain
variable region comprising a CDRHi comprising the amino acid sequence of SEQ
ID NO: 250, a
CDR112 comprising the amino acid sequence of SEQ ID NO: 251, and a CDR113
comprising the
amino acid sequence of SEQ ID NO: 163, wherein CDRiii, CDRH2, and CDRH3
sequences are
interposed between immunoglobulin FR sequences (PAL769-VH); and/or an
immunoglobulin
light chain variable region comprising a CDRIA comprising the amino acid
sequence of SEQ ID
NO: 253, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 254, and a
CDRL3
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comprising the amino acid sequence of SEQ ID NO: 166, wherein the CDRL1,
CDRL2, and
CDRL3 sequences are interposed between immunoglobulin FR sequences (PAL769-
VL).
[0129]
In certain embodiments, an antibody can comprise: an immunoglobulin heavy
chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 250, a
CDRH2 comprising the amino acid sequence of SEQ ID NO: 252, and a CDRH3
comprising the
amino acid sequence of SEQ ID NO: 163, wherein CDRH1, CDR112, and CDRH3
sequences are
interposed between immunoglobulin FR sequences (h769-VH); and/or an
immunoglobulin light
chain variable region comprising a CDRL1 comprising the amino acid sequence of
SEQ ID NO:
255, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 254, and a CDRL3
comprising the amino acid sequence of SEQ ID NO: 166, wherein the CDRL1,
CDRL2, and
CDRL3 sequences are interposed between immunoglobulin FR sequences (h769-IF3-
VL, h769-
tm2-VL, h769-tm3-VL).
[0130] In certain embodiments, an antibody can comprise: an immunoglobulin
heavy chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 161,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 162, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 163, wherein CDRH1, CDRH2, and CDRH3
sequences
are interposed between immunoglobulin FR sequences (PAL769-VH, h769-VH);
and/or an
immunoglobulin light chain variable region comprising a CDRL1 comprising the
amino acid
sequence of SEQ ID NO: 165, a CDRL2 comprising the amino acid sequence of SEQ
ID NO:
142, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 203, wherein
the
CDRIA, CDR-Li., and CDRL3 sequences are interposed between immunoglobulin FR
sequences
(h769.T-VL).
[0131] In certain embodiments, an antibody can comprise: an immunoglobulin
heavy chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 250, a
CDR42 comprising the amino acid sequence of SEQ ID NO: 252, and a CDRH3
comprising the
amino acid sequence of SEQ ID NO: 163, wherein CDRHI, CDRH2, and CDRH3
sequences are
interposed between immunoglobulin FR sequences (h769-VH); and/or an
immunoglobulin light
chain variable region comprising a CDRL1 comprising the amino acid sequence of
SEQ ID NO:
255, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 254, and a CDRL3
comprising the amino acid sequence of SEQ ID NO: 203, wherein the CDRL1,
CDRL2, and
CDRL3 sequences are interposed between immunoglobulin FR sequences (h769.T-
VL).
101321 In certain embodiments, an antibody can comprise: an immunoglobulin
heavy chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 129,
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a CDRH2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 131, wherein CDRH1, CDRH2, and CDRH3
sequences
are interposed between immunoglobulin FR sequences (PAL752-VH); and/or an
immunoglobulin light chain variable region comprising a CDRL1 comprising the
amino acid
sequence of SEQ ID NO: 133, a CDRL2 comprising the amino acid sequence of SEQ
ID NO:
134, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 135, wherein
the
CDRL1, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR
sequences
(PAL752-VL).
101331 In certain embodiments, an antibody can comprise: an immunoglobulin
heavy chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 137,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 138, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 139, wherein CDRH1, CDRH2, and CDRH3
sequences
are interposed between immunoglobulin FR sequences (PAL759-VH); and/or an
immunoglobulin light chain variable region comprising a CDRL1 comprising the
amino acid
sequence of SEQ ID NO: 141, a CDRL2 comprising the amino acid sequence of SEQ
ID NO:
142, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 143, wherein
the
CDRL1, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR
sequences
(PAL759-VL).
101341 In certain embodiments, an antibody can comprise: an immunoglobulin
heavy chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 145,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 147, wherein CDRHi, CDRH2, and CDRH3
sequences
are interposed between immunoglobulin FR sequences (PAL760-VH); and/or an
immunoglobulin light chain variable region comprising a CDRL1 comprising the
amino acid
sequence of SEQ ID NO: 149, a CDRL2 comprising the amino acid sequence of SEQ
ID NO:
150, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 151, wherein
the
CDRL1, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR
sequences
(PAL760-VL).
101351 In certain embodiments, an antibody can comprise: an immunoglobulin
heavy chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 153,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 154, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 155, wherein CDRH1, CDRH2, and CDRH3
sequences
are interposed between immunoglobulin FR sequences (PAL767-VH); and/or an
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immunoglobulin light chain variable region comprising a CDRL1 comprising the
amino acid
sequence of SEQ ID NO: 157, a CDRL2 comprising the amino acid sequence of SEQ
ID NO:
158, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 159, wherein
the
CDRL1, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR
sequences
(PAL767-VL).
101361 In certain embodiments, an antibody can comprise: an immunoglobulin
heavy chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 161,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 168, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 169, wherein CDRH1, CDRH2, and CDRH3
sequences
are interposed between immunoglobulin FR sequences (PAL771-VH); and/or an
immunoglobulin light chain variable region comprising a CDRL1 comprising the
amino acid
sequence of SEQ ID NO: 171, a CDRL2 comprising the amino acid sequence of SEQ
ID NO:
172, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 173, wherein
the
CDRL1, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR
sequences
(PAL771-VL).
101371 In certain embodiments, an antibody can comprise: an immunoglobulin
heavy chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 175,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 176, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 177, wherein CDRHi, CDRH2, and CDRH3
sequences
are interposed between immunoglobulin FR sequences (PAL785-VH); and/or an
immunoglobulin light chain variable region comprising a CDRL1 comprising the
amino acid
sequence of SEQ ID NO: 179, a CDRL2 comprising the amino acid sequence of SEQ
ID NO:
180, and a CDRL3 comprising the amino acid sequence of SEQ 11) NO: 181,
wherein the
CDRL1, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR
sequences
(PAL785-VL).
101381 In certain embodiments, an antibody can comprise: an immunoglobulin
heavy chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 183,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 184, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 185, wherein CDRH1, CDRH2, and CDRH3
sequences
are interposed between immunoglobulin FR sequences (PAL787-VH); and/or an
immunoglobulin light chain variable region comprising a CDRL1 comprising the
amino acid
sequence of SEQ ID NO: 187, a CDRL2 comprising the amino acid sequence of SEQ
ID NO:
188, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 189, wherein
the
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CDRIA, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR
sequences
(PAL787-VL).
[0139] In certain embodiments, an antibody can comprise: an immunoglobulin
heavy chain
variable region comprising a CDRH1 comprising the amino acid sequence of SEQ
ID NO: 191,
a CDRH2 comprising the amino acid sequence of SEQ ID NO: 192, and a CDRH3
comprising
the amino acid sequence of SEQ ID NO: 193, wherein CDRHi, CDRH2, and CDRH3
sequences
are interposed between immunoglobulin FR sequences (PAL788-V11); and/or an
immunoglobulin light chain variable region comprising a CDRL1 comprising the
amino acid
sequence of SEQ ID NO: 195, a CDRL2 comprising the amino acid sequence of SEQ
ID NO:
196, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 197, wherein
the
CDRIA, CDRL2, and CDRL3 sequences are interposed between immunoglobulin FR
sequences
(PAL788-VL).
[0140] Similarly, the antibodies disclosed herein can comprise an
immunoglobulin heavy chain
variable region and an immunoglobulin light chain variable region.
[0141] In certain embodiments, the antibody comprises an immunoglobulin heavy
chain variable
region comprising an amino acid sequence selected from SEQ ID NO: 164, SEQ ID
NO: 199,
SEQ ID NO: 132, SEQ ID NO: 140, SEQ ID NO: 148, SEQ ID NO: 156, SEQ ID NO:
170,
SEQ ID NO: 178, SEQ ID NO: 186, and SEQ ID NO: 194; and/or an immunoglobulin
light
chain variable region comprising an amino acid sequence selected from SEQ ID
NO: 167, SEQ
ID NO. 200, SEQ ID NO. 201, SEQ ID NO. 202, SEQ ID NO. 204, SEQ ID NO. 136,
SEQ ID
NO: 144, SEQ ID NO: 152, SEQ ID NO: 160, SEQ ID NO: 174, SEQ ID NO: 182, SEQ
ID NO:
190, SEQ ID NO: 198, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID
NO: 245,
SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248 and SEQ ID NO: 249.
[0142] In certain embodiments, the antibody comprises an immunoglobulin heavy
chain variable
region comprising the amino acid sequence of SEQ ID NO: 164 (PAL769-VH), and
an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
167 (PAL769-VL).
[0143] In certain embodiments, the antibody comprises an immunoglobulin heavy
chain variable
region comprising the amino acid sequence of SEQ ID NO: 199 (h769 VII), and an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
200 (h769-IF3-VL).
[0144] In certain embodiments, the antibody comprises an immunoglobulin heavy
chain variable
region comprising the amino acid sequence of SEQ ID NO: 199 (h769 VII), and an
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immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
201 (h769-tm2-VL).
101451 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain variable
region comprising the amino acid sequence of SEQ ID NO: 199 (h769 VII), and an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
202 (h769-tm3-VL).
101461 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain variable
region comprising the amino acid sequence of SEQ ID NO: 199 (h769 VII), and an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
204 (h769.T-VL).
101471 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain variable
region comprising the amino acid sequence of SEQ ID NO: 132 (P4L752-VH), and
an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
136 (PAL752-VL).
101481 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain variable
region comprising the amino acid sequence of SEQ ID NO: 140 (PAL759-VH), and
an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
144 (PAL759-VL).
101491 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain variable
region comprising the amino acid sequence of SEQ ID NO: 148 (PAL760-VH), and
an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
152 (PAL760-VL).
101501 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain variable
region comprising the amino acid sequence of SEQ ID NO: 156 (PAL767-VH), and
an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
160 (PAL767-VL).
101511 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain variable
region comprising the amino acid sequence of SEQ ID NO: 170 (PAL771-VH), and
an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
174 (PAL771-VL).
101521 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain variable
region comprising the amino acid sequence of SEQ ID NO: 178 (PAL785-VH), and
an
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immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
182 (PAL785-VL).
101531 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain variable
region comprising the amino acid sequence of SEQ ID NO: 186 (PAL787-VH), and
an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
190 (PAL787-VL).
101541 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain variable
region comprising the amino acid sequence of SEQ ID NO: 194 (PAL788-VH), and
an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
198 (PAL788-VL).
101551 In certain embodiments the antibody comprises an immunoglobulin heavy
chain variable
region comprising an amino acid sequence of
EVQLX1X2SGAEX3X4KP GAX5VX6X7S CX8X9S G FN I KDTYMHWVXi 0QX1IPX12X13GLEWX14GX15
I DPANDNIX16YX17X18KFQX19X20X22T I TADTSX22DTAYX23X24LSSLX25SEDTAVYYCAREGY
GGSYGEGYWGQGTX28X27TVSS (SEQ ID NO: 236),
wherein Xi is Gln or Val, X2 is Glu or Gln, X3 is Leu or Val, X4 is Val or
Lys, X5 is Ser or Thr,
X6 is Thr or Lys, X7 is Leu or Ile, Xs is Thr or Lys, X9 is Ala or Val, Xio is
Lys or Gln, Xii is Arg
or Ala, X12 is Glu or Gly, X13 is Gln or Lys, X14 is Ile or Met, X15 is Arg or
Leu, X16 is Lys or Ile,
X17 is Asp or Ala, X18 is Pro or Glu, X19 is Asp or Gly, X2ois Lys or Arg, X21
is Ala or Val, X22is
Ser or Thr, X23 is Leu or Met, X24 is Arg or Glu, X25 is Thr or Arg, X26is Thr
or Leu, and X27 is
Leu or Val; and/or an immunoglobulin light chain variable region comprising an
amino acid
sequence of
KiIVmTQX2PX3X4LX5X6SX7GX0RvTX9XioCXIIASQSVsNDX12X13WYQQKPGQX14PX1.5LL I YY
AS IRFTGX18PX17RFX18GSGX19GTDFTX20T IX21X22X23QX24EDX25AVYX26CQQDYX27 SPWT F
GX28GTKX29E (SEQ ID NO: 237),
wherein Xi is Ser or Glu, X7 is Thr or Ser, X3 is Lys or Pro, X4 is Phe or
Thr, X5 is Leu or Ser, X6
is Val or Leu, X7 is Ala or Pro, Xs is Asp or Glu, X9 is Ile or Leu, Xio is
Thr or Ser, Xii is Lys or
Arg, X12 is Val or Leu, X13 is Ile or Ser, X14 is Ser or Ala, X15 is Lys or
Arg, X16 is Val or Ile, X17
is Asp or Ala, X18 is Ala or Ser, X19 is Tyr or Ser, X70 is Phe or Leu, X71 is
Asn or Ser, X22 is Thr
or Ser, X23 is Val or Leu, X24 is Ala or Pro, X25 is Leu or Phe, XTh is Phe or
Tyr, X77 is Tyr or Thr,
Xis is Gly or Gln, and X29 is Leu or Val.
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101561 In certain embodiments, an isolated antibody that binds PD-Li comprises
an
immunoglobulin heavy chain variable region comprising an amino acid sequence
that is at least
70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 89.5%, 90%, 90.5%, 91%, 92%, 93%, 94%,
95%,
96%, 97%, 98%, or 99% identical to the entire variable region and/or the
framework region
sequences of an amino acid sequence selected from SEQ ID NO: 164, SEQ ID NO:
199, SEQ ID
NO: 132, SEQ ID NO: 140, SEQ ID NO: 148, SEQ ID NO: 156, SEQ ID NO: 170, SEQ
ID NO:
178, SEQ ID NO: 186, and SEQ ID NO: 194. Alternatively or in addition, an
isolated antibody
that binds PD-Li comprises an immunoglobulin light chain variable region
comprising an amino
acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%,
93%, 94%, 94.5%, 95%, 95.5%, 96%, 97%, 98%, or 99% identical to the entire
variable region
and/or the framework region sequences of an amino acid sequence selected from
SEQ ID NO:
167, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID
NO: 136,
SEQ ID NO: 144, SEQ ID NO: 152, SEQ ID NO: 160, SEQ ID NO: 174, SEQ ID NO:
182,
SEQ ID NO: 190, SEQ ID NO: 198, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO:
244,
SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248 and SEQ ID NO:
249.
Identification of CDR and framework sequences is within the level of ordinary
skill in the art,
and it is understood that the boundaries between CDR and framework sequences
may depend
upon the definition or convention that is used (e.g., Kabat, Chothia, EVIGT,
etc.). Exemplary
CDR and framework sequences for heavy chain variable region sequences SEQ ID
NO: 164 and
SEQ ID NO: 199 are depicted in FIGURE 51A, and exemplary CDR and framework
sequences
for light chain variable region sequences SEQ ID NO: 167, SEQ ID NO: 200, SEQ
ID NO: 201,
SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO:
244,
SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, and SEQ ID NO:
249
are depicted in FIGURE 51B.
101571 Sequence identity may be determined in various ways that are within the
skill in the art,
e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN
or
Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool)
analysis using
the algorithm employed by the programs blastp, blastn, blastx, tblastn and
tblastx (Karlin et al.,
(1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul, (1993) J. MoL. EvoL.
36, 290-
300; Altschul et al., (1997) NUCLEIC ACIDS RES. 25:3389-3402, incorporated by
reference) are
tailored for sequence similarity searching. For a discussion of basic issues
in searching sequence
databases, see Altschul etal., (1994) NATURE GENETICS 6:119-129, which is
fully incorporated
by reference. Those skilled in the art can determine appropriate parameters
for measuring
alignment, including any algorithms needed to achieve maximal alignment over
the full length of
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the sequences being compared. The search parameters for histogram,
descriptions, alignments,
expect (i.e., the statistical significance threshold for reporting matches
against database
sequences), cutoff, matrix and filter are at the default settings. The default
scoring matrix used
by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et
al., (1992) PROC.
NATL. ACAD. Scf. USA 89:10915-10919, fully incorporated by reference). Four
blastn
parameters may be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap
extension
penalty); wink=1 (generates word hits at every winkth position along the
query); and
gapw=16 (sets the window width within which gapped alignments are generated).
The
equivalent Blastp parameter settings may be Q=9; R=2; wink=1; and gapw=32.
Searches may
also be conducted using the NCBI (National Center for Biotechnology
Information) BLAST
Advanced Option parameter (e.g: -G, Cost to open gap [Integer]: default = 5
for nucleotides/ 11
for proteins; -E, Cost to extend gap [Integer]: default = 2 for nucleotides/ 1
for proteins; -q,
Penalty for nucleotide mismatch [Integer]: default ¨ -3; -r, reward for
nucleotide match
[Integer]: default = 1; -e, expect value [Real]: default = 10; -W, wordsize
[Integer]: default = 11
for nucleotides/ 28 for megablast/ 3 for proteins; -y, Dropoff (X) for blast
extensions in bits:
default = 20 for blastn/ 7 for others; -X, X dropoff value for gapped
alignment (in bits): default =
15 for all programs, not applicable to blastn; and ¨Z, final X dropoff value
for gapped alignment
(in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein
alignments may also be
used (default parameters may include, e.g., Blosum62 matrix and Gap Opening
Penalty = 10 and
Gap Extension Penalty = 0.1). A Bestfit comparison between sequences,
available in the GCG
package version 10.0, uses DNA parameters CiAP=50 (gap creation penalty) and
LEN=3 (gap
extension penalty) and the equivalent settings in protein comparisons are
GAP=8 and LEN=2.
101581 In each of the foregoing embodiments, it is contemplated herein that
immunoglobulin
heavy chain variable region sequences and/or light chain variable region
sequences that together
bind PD-Li may each contain amino acid alterations (e.g, at least 1, 2, 3, 4,
5, or 10 amino acid
substitutions, deletions, or additions) in the framework regions of the heavy
and/or light chain
variable regions.
101591 In certain embodiments, it is contemplated that a heavy chain variable
region sequence,
for example, the VH sequence of SEQ ID NO: 164, SEQ ID NO: 199, SEQ ID NO:
132, SEQ ID
NO: 140, SEQ ID NO: 148, SEQ ID NO: 156, SEQ lD NO: 170, SEQ lD NO: 178, SEQ
ID NO:
186, or SEQ ID NO: 194, or the amino acid variants thereof, may be covalently
linked to a
variety of heavy chain constant region sequences known in the art. Similarly,
it is contemplated
that a light chain variable region sequence, for example, the VI_ of SEQ ID
NO: 167, SEQ ID
NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 204, SEQ lD NO: 136, SEQ
lD NO:
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144, SEQ ID NO: 152, SEQ ID NO: 160, SEQ ID NO: 174, SEQ ID NO: 182, SEQ ID
NO: 190,
SEQ ID NO: 198, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO:
245,
SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248 or SEQ ID NO: 249, or the amino
acid
variants thereof, may be covalently linked to a variety of light chain
constant region sequences
known in the art.
[0160] For example, the antibody molecule may have a heavy chain constant
region chosen
from, e.g., the heavy chain constant regions of IgGl, IgG2, IgG3, IgG4, IgM,
IgAl, IgA2, IgD,
and IgE; particularly, chosen from, e.g., the (e.g., human) heavy chain
constant regions of IgGl,
IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has a light
chain constant
region chosen from, e.g., the (e.g., human) light chain constant regions of
kappa or lambda. The
constant region can be altered, e.g., mutated, to modify the properties of the
antibody (e.g., to
increase or decrease one or more of: Fc receptor binding, antibody
glycosylation, the number of
cysteine residues, effector cell function, and/or complement function). In one
embodiment the
antibody has effector function and can fix complement. In other embodiments
the antibody does
not recruit effector cells or fix complement. In another embodiment, the
antibody has reduced or
no ability to bind an Fc receptor. For example, it is an isotype or subtype,
fragment or other
mutant, which does not support binding to an Fc receptor, e.g., it has a
mutagenized or deleted
Fc receptor binding region.
[0161] In certain embodiments, the constant region of the heavy chain of the
antibody is a
human IgG1 isotype, having an amino acid sequence:
AS TKGPSVFPLAPSSKS T S GGTAALGCLVKDYFPEPVTVSWNSGALT GVHT FPAVLQS S
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
P SVFL FPPKPKDTLMI SRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYE
S TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT I SKAKGQPREPQVYTLPPSRDE
L TKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGS FFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 227).
101621 In certain embodiments, the human IgG1 constant region is modified at
amino acid
Asn297 (boxed in SEQ ID NO: 227 in the preceding paragraph) to prevent to
glycosylati on of
the antibody, for example Asn297Ala (N297A) or Asn297Gly (N297G). In certain
embodiments, the constant region of the antibody is modified at amino acid
Leu235 (boxed in
SEQ ID NO: 227 in the preceding paragraph) to alter Fc receptor interactions,
for example
Leu235Glu (L235E) or Leu235Ala (L235A). In certain embodiments, the constant
region of the
antibody is modified at amino acid Leu234 (boxed in SEQ ID NO: 227 in the
preceding
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paragraph) to alter Fc receptor interactions, e.g., Leu234A1a (L234A). In
certain embodiments,
the constant region of the antibody is modified at amino acid Glu233 (boxed in
SEQ ID NO: 227
in the preceding paragraph), e.g., Glu233Pro (E233P). In certain embodiments,
the constant
region of the antibody is altered at both amino acid 234 and 235, for example
Leu234Ala and
Leu235Ala (L234A/L235A). In certain embodiments, the constant region of the
antibody is
altered at amino acids 233, 234, and 235, for example, Glu233Pro, Leu234A1a,
and Leu235Ala
(E233P L234A/L235A) (Armour KL. et at. (1999) EUR. J. ImmuNoL. 29(8):2613-24).
In certain
embodiments, the constant region of the antibody is altered at amino acids
234, 235 and 329, for
example, Leu234Ala, Leu235Ala and Pro329Gly. (see, e.g., U.S. Patent No.
8,969,526). All
residue numbers are according to EU numbering (Kabat, E.A., et al., supra).
[0163] In certain embodiments, the constant region of the heavy chain of the
antibody is a
human IgG1 isotype, having an amino acid sequence:
AS TKGPSVFPLAPS SKS T S GGTAALGCLVKDYFPE PVTVSWNS GALT S GVHT FPAVLQS S
GLYS LS SVVTVPS S SLGTQT Y ICNVNHKP SNTKVDKKVE PKS CDKTHT C PPCPAPELL GG
PSVFLFJ7PKPKDTLMI SRT PEVT CVVVDVSHE DPEVKFNWYVDGVEVHNAKTKDREE QYE
S TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKT I SKAKGQPREPQVYTLPPSREE
MTKNQVSL TCLVKGFYPSD IAVEWESNGQPENNYKT TPPVLDSDGS FFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYT QKS LS LS PGK (SEQ ID NO: 221).
[0164] In certain embodiments, the human IgG1 constant region is modified at
amino acid
Asn297 (boxed in SEQ ID NO: 221 the preceding paragraph) to prevent to
glycosylation of the
antibody, for example Asn297Ala (N297A) or Asn297Gly (N297G). For example, in
certain
embodiments, the human IgG1 constant region comprises SEQ ID NO: 222, SEQ ID
NO: 225,
or SEQ ID NO: 226. In certain embodiments, the constant region of the antibody
is modified at
amino acid Leu235 (boxed in SEQ ID NO: 221 the preceding paragraph) to alter
Fc receptor
interactions, for example Leu235Glu (L235E) or Leu235Ala (L235A). In certain
embodiments,
the constant region of the antibody is modified at amino acid Leu234 (boxed in
SEQ ID NO: 221
the preceding paragraph) to alter Fc receptor interactions, e.g., Leu234Ala
(L234A). In certain
embodiments, the constant region of the antibody is modified at amino acid
Glu233 (boxed in
SEQ ID NO: 221 the preceding paragraph), e.g., Glu233Pro (E233P). In certain
embodiments,
the constant region of the antibody is altered at both amino acid 234 and 235,
for example
Leu234Ala and Leu235Ala (L234A/L235A). In certain embodiments, the constant
region of the
antibody is altered at amino acids 233, 234, and 234, for example, Glu233Pro,
Leu234A1a, and
Leu235Ala (E233P L234A/L235A) (Armour KL. et at. (1999) EUR. J. ImivruNoL.
29(8):2613-
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24). In certain embodiments, the constant region of the antibody is altered at
amino acids 234,
235 and 329, for example, Leu234A1a, Leu235Ala and Pro329Gly. (see, e.g., U.S.
Patent No.
8,969,526). All residue numbers are according to EU numbering (Kabat, E.A., et
at., supra).
101651 In certain embodiments, the human IgG1 constant region is modified to
comprise either
a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, for
heterodimerization with a
second constant region (residue numbers according to EU numbering (Kabat,
E.A., et al.,
supra)). For example, in certain embodiments, the human IgG1 constant region
comprises a
Y407T mutation (e.g., the human IgG1 constant region comprises SEQ ID NO: 223
or SEQ ID
NO: 225). In certain embodiments, the human IgG1 constant region comprises a
T366Y
mutation (e.g., the human IgG1 constant region comprises SEQ ID NO: 224 or SEQ
ID NO:
226).
101661 In certain embodiments, the constant region of the heavy chain of the
antibody is a
human IgG1 isotype, e.g., an allotype of the human IgG1 isotype, e.g., the
IgG1 G1m3 allotype.
Exemplary human IgG1 allotypes are described in Magdelaine-Beuzelin et at.
(2009)
PIIARMACOGENET. GENOMICS 19(5):383-7.
101671 In certain embodiments, the constant region of the heavy chain of the
antibody is a
human IgG2 isotype, having an amino acid sequence:
AS TKGPSVFPLAPCSRS TSES TAALGCLVKDYFPE PVTVSWNS GALT S GVHT FPAVLQS S
GLYS LS SVVTVPS SNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF
L FP PKPKDT LMI S RT PEVT CVVVDVS HE D PEVQ FNWYVDGVEVHNAKT KPREEQFN S T FR
VVSVLIVVHQPWLNGKEYKCKVSNKGLPAP IEKT I SKTKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDI SVEWE SNGQPENNYKT TPPMLDS DGS FFLYSKL TVDKSRWQQGN
VFS CSVMHEALHNHYTQKS L S LS PGK (SEQ ID NO: 228).
101681 In certain embodiments, the human IgG-2 constant region is modified at
amino acid
Asn297 (boxed in SEQ ID NO: 228 in the preceding paragraph) to prevent to
glycosylation of
the antibody, e.g., A.sn297AI a (N297A) or Asn297Gly (N297G), where the
residue numbers are
according to EU numbering (Kabat, E.A., et at., supra).
101691 In certain embodiments, the constant region of the heavy chain of the
antibody is an
human IgG3 isotype, having an amino acid sequence:
AS TKGPSVFPLAPCSRS T S GGTAALGCLVKDYFPE PVTVSWNS GALT S GVHT FPAVLQS S
GLYS LS SVVTVPS S SLGTQT Y TCNVNHKP SNTKVDKRVE LKT PLGDT THTCPRCPE PKS C
DT PPPCPRCPE PKSCDT PPPC PRCPE PKS CDT PPPCPRC PAPELLGGP SVFL FPPKPKDT
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LM I S RT PEVT CVVVDVS HE D PEVQ FKWYVDGVEVHNAKT KPREE QYNS T FRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAP IEKT I SKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPS D IAVEWE S SGQPENNYNT T PPMLDS DGS FFLYSKL TVDKSRWQQGNI FS CSVMHE
ALHNEIFTQKS L S LS PGK (SEQ ID NO: 229).
[0170] In certain embodiments, the human 1gG3 constant region is modified at
amino acid
Asn297 (boxed in SEQ ID NO: 229 in the preceding paragraph) to prevent to
glycosylation of
the antibody, e.g.., Asn297A1a (N297A) or Asn297G1y (N297G). In certain
embodiments, the
human IgG3 constant region is modified at amino acid Arg435 (boxed in SEQ ID
NO: 229 in the
preceding paragraph) to extend the half-life, e.g., Arg435H (R435H). All
residue numbers are
according to EU numbering (Kabat, EA., et al., supra).
[0171] In certain embodiments, the constant region of the heavy chain of the
antibody is an
human IgG4 isotype, having an amino acid sequence:
AS TKGPSVFPLAPCSRS TSES TAALGCLVKDYFPE PVTVSWNS GALT S GVHT FPAVLQS S
GLYS LS SVVTVPS S S LGTKT Y TCNVDHKP SNTKVDKRVE S KYGP PCPEC PAPE FL GGPSV
FL FPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVENAKTKPREEQFFTS TY
RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKT I SKAKGQPREPQVYTLPPSQEEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEG
NVFSCSVMHEALI-INHYTQKSLSLSLGK (SEQ ID NO: 230).
[0172] In certain embodiments, the human IgG4 consta.nt region is modified.
within the hinge
region to prevent or reduce strand exchange, e.g., in certain embodiments
human IgG4 constant
region is modified at Ser228 (boxed in SEQ ID NO: 230 in the preceding
paragraph), e.g.,
Ser228Pro (S228P). In other embodiments, the human IgG4 constant region is
modified at
amino acid Leu235 (boxed in SEQ ID NO: 230 in the preceding paragraph) to
alter Fe receptor
interactions, e.g., Len235Giu (L235E). In certain embodiments, the human IgG4
constant region
is modified at both Ser228 and Leu335, e.g., Ser228Pro and Leu235G1u.
(S228P/L235E). In
certain embodiments, the human IgG4 constant region is modifie,d at amino acid
Asn297 (boxed
in SEQ ID NO: 230 in the preceding paragraph) to prevent to glycosylation of
the antibody, e.g.,
Asn297Ala (N297A) or Asn297Gly (N297G). All residue numbers are according to
EU
numbering (Kabat, E.A., et al., supra).
[0173] In certain embodiments, the human IgG constant region is modified to
enhance FoRn
binding. Examples of Fe mutations that enhance binding to FeRn are
rtilet252Tyr, Ser254Thr,
Thr256Giu (M252Y, S254T, 17256E, respectively) (Dall'Acqua et al. (2006) J.
BIOL. CHEM.
281(33): 23514-23524), or Met428Leu and A.sn434Ser (1144281õ N434S) (Zalevsky
et al. (2010)
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NATURE BiunicH. 28(2): 157-159). All residue numbers are according to EU
numbering (Kabat,
E.A., et al., supra).
101741 Tn certain embodiments, the human IgG constant region is modified to
alter antibody-
dependent cellular cytotoxicity (ADCC) and/or complement-dependent
cytotoxicity (CDC), e.g.,
the amino acid modifications described in Natsume cal. (2008) CANCER RES.
68(10): 3863-72;
Ildusogie et al. (2001) J. ImmuNOL. 166(4): 2571-5; Moore etal. (2010) NIABs
2(2): 181-189;
Lazar et al.. (2006) PROC. NATL. ACAD. SCT. USA 103(11): 4005-4010, Shields
etal. (2001) J.
BIOL. CHEM. 276(9): 6591-6604; Stavenhagen etal. (2007) CANCER RES. 67(18):
8882-8890;
Stavenhage.n etal. (2008) .ADVAN. ENZYME REGUL, 48: 152-164; Alegre etal.
(1992) J.
ImMUNOL. 148: 3461-3468.
101751 In certain embodiments, the human Ig-Ci constant region is modified to
induce
heterodimerization. For example, a heavy chain having an amino acid
modification within the
CEP domain at Thr366, e.g., a substitution with a more bulky amino acid, e.g.,
Tyr (T366W), is
able to preferentially pair with a second heavy chain having a (71-1-3 domain
having, amino acid
modifications to less bulky amino acids at positions Thr366, Leu368, and
1yr407, e.g., Ser, Ala
and Val, respectively (T366S/L368A/Y407V). Eleterodimerization via CI-I3
modifications can
be further stabilized by the introduction of a disulfide bond, for example by
changing Ser354 to
Cys (S354C) and Y349 to Cys (X349C) on opposite CE13 domains (see, Carter
(2001) J.
IMMUNOL. METHODS 248: 7-15).
101761 In certain embodiments, the constant region of the light chain of the
antibody is a human
kappa constant region, e.g., a human kappa constant region having the amino
acid sequence.
TVAAPSVFI FP PSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQS GNS QESVTEQDSKDS T
YSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEO ID NO: 231),
101771 In certain embodiments, the constant region of the light chain of the
antibody is a human
kappa constant region, e.g., a human kappa constant region having the amino
acid sequence.
RTVAAPSVFI FPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQS GNS QESVTE QDSKDS
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVIKSFNRGEC (SEC) ED NO: 232).
101781 In certain embodiments, the constant region of the light chain of the
antibody is a human
lambda constant region, e.g., a human lambda constant region having the amino
acid sequence:
GQPKANPTVTL FPPSSEELQANKATLVCL I SDFYPGAVTVAWKADGS PVKAGVET TKP SKQSNN
KYAAS SYLS L T PEQWKSHRS YSCQVTHEGS TVEKTVAPTEC (SEQ ID NO: 233).
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101791 In certain embodiments, the antibody comprises an immunoglobulin heavy
chain
comprising the amino acid sequence of SEQ ID NO: 235, or an amino acid
sequence that has at
least 85%, 90%, 95%, 95.5%, 96%, 96.5%, 97%, 98%, or 99% sequence identity to
SEQ ID NO:
235; and/or an immunoglobulin light chain comprising the amino acid sequence
of SEQ ID NO:
205, or an amino acid sequence that has at least 85%, 90%, 94.5% 95%, 95.5%,
96%, 97%, 98%,
or 99% sequence identity to SEQ ID NO: 205.
101801 In certain embodiments, the antibody binds human PD-Li with a Ku of 20
nM, 15 nM,
nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1
nM,
0.075 nM, or 0.05 nM or lower, as measured using standard binding assays, for
example, surface
10 plasmon resonance or bio-layer interferometry. In certain embodiments,
the antibody binds
human PD-Li with a KD of from about 20 nM to about 0.05 nM, from about 20 nM
to about
0.075 nM, from about 20 nM to about 0.1 nM, from about 20 nM to about 0.5 nM,
from about
nM to about 1 nM, from about 10 nM to about 0.05 nM, from about 10 nM to about
0.075
nM, from about 10 nM to about 0.1 nM, from about 10 nM to about 0.5 nM, from
about 10 nM
15 to about 1 nM, from about 5 nM to about 0.05 nM, from about 5 nM to
about 0.075 nM, from
about 5 nM to about 0.1 nM, from about 5 nM to about 0.5 nM, from about 5 nM
to about 1 nM,
from about 3 nM to about 0.05 nM, from about 3 nM to about 0.075 nM, from
about 3 nM to
about 0.1 nM, from about 3 nM to about 0.5 nM, from about 3 nM to about 1 nM,
from about 3
nM to about 2 nM, from about 2 nM to about 0.05 nM, from about 2 nM to about
0.075 nM,
20 from about 2 nM to about 0.1 nM, from about 2 nM to about 0.5 nM, from
about 2 nM to about
1 nM, from about 1 nM to about 0.05 nM, from about 1 nM to about 0.075 nM,
from about 1 nM
to about 0.1 nM, from about 1 nM to about 0.5 nM, from about 0.5 nM to about
0.05 nM, from
about 0.5 nM to about 0.075 nM, from about 0.5 nM to about 0.1 nM, from about
0.1 nM to
about 0.05 nM, from about 0.1 nM to about 0.075 nM, or from about 0.075 nM to
about 0.05
nM, or from about 0.05 nM to about 0.035 nM, as measured using standard
binding assays, for
example, surface plasmon resonance or bio-layer interferometry.
101811 In certain embodiments, in addition to binding human PD-L1, a disclosed
antibody also
binds to Macaca fascicularis (cynomolgus) PD-Li. For example, the antibody
binds
cynomolgus PD-Li with a KD of 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5
nM, 4 nM,
3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.075 nM, or 0.05 nM or lower, as
measured using
standard binding assays, for example, surface plasmon resonance or bio-layer
interferometry. In
certain embodiments, the antibody binds cynomolgus PD-Li with a Ku of from
about 20 nM to
about 0.05 nM, from about 20 nM to about 0.075 nM, from about 20 nM to about
0.1 nM, from
about 20 nM to about 0.5 nM, from about 20 nM to about 1 nM, from about 10 nM
to about 0.05
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nM, from about 10 nM to about 0.075 nM, from about 10 nM to about 0.1 nM, from
about 10
nM to about 0.5 nM, from about 10 nM to about 1 nM, from about 5 nM to about
0.05 nM, from
about 5 nM to about 0.075 nM, from about 5 nM to about 0.1 nM, from about 5 nM
to about 0.5
nM, from about 5 nM to about 1 nM, from about 3 nM to about 0.05 nM, from
about 3 nM to
about 0.075 nM, from about 3 nM to about 0.1 nM, from about 3 nM to about 0.5
nM, from
about 3 nM to about 1 nM, from about 3 nM to about 2 nM, from about 2 nM to
about 0.05 nM,
from about 2 nM to about 0.075 nM, from about 2 nM to about 0.1 nM, from about
2 nM to
about 0.5 nM, from about 2 nM to about 1 nM, from about 1 nM to about 0.05 nM,
from about 1
nM to about 0.075 nM, from about 1 nM to about 0.1 nM, from about 1 nM to
about 0.5 nM,
from about 0.5 nM to about 0.05 nM, from about 0.5 nM to about 0.075 nM, from
about 0.5 nM
to about 0.1 nM, from about 0.1 nM to about 0.05 nM, from about 0.1 nM to
about 0.075 nM, or
from about 0.075 nM to about 0.05 nM, as measured using standard binding
assays, for example,
surface plasmon resonance or bio-layer interferometry.
101821 In certain embodiments, the antibody interferes with the binding of PD-
Li to PD-1.
101831 In certain embodiments, the invention provides antibodies that bind to
the same epitope
present in PD-Li as that bound by an antibody disclosed herein. In certain
embodiments, the
invention provides antibodies that compete for binding to PD-Li with an
antibody disclosed
herein.
101841 Competition assays for determining whether an antibody binds to the
same epitope as, or
competes for binding with a disclosed antibody are known in the art. Exemplary
competition
assays include immunoassays (e.g., ELISA assays, RIA assays), surface plasmon
resonance,
(e.g., BIAcore analysis), bio-layer interferometry, and flow cytometry.
101851 Typically, a competition assay involves the use of an antigen (e.g., a
human PD-Li.
protein or fragment thereof) bound to a solid surface or expressed on a cell
surface, a test PD-
LI-binding antibody and a reference antibody. The reference antibody is
labeled and the test
antibody is unlabeled. Competitive inhibition is measured by determining the
amount of labeled
reference antibody bound to the solid surface or cells in the presence of the
test antibody.
Usually the test antibody is present in excess (e.g., lx, 5x, 10x, 20x or
100x). Antibodies
identified by competition assay (i.e., competing antibodies) include
antibodies binding to the
same epitope, or similar (e.g., overlapping) epitopes, as the reference
antibody, and antibodies
binding to an adjacent epitope sufficiently proximal to the epitope bound by
the reference
antibody for steric hindrance to occur.
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101861 A competition assay can be conducted in both directions to ensure that
the presence of
the label does not interfere or otherwise inhibit binding. For example, in the
first direction the
reference antibody is labeled and the test antibody is unlabeled, and in the
second direction, the
test antibody is labeled and the reference antibody is unlabeled.
101871 A test antibody competes with the reference antibody for specific
binding to the antigen
if an excess of one antibody (e.g., lx, 5x, 10x, 20x or 100x) inhibits binding
of the other
antibody, e.g., by at least 50%, 75%, 90%, 95% or 99% as measured in a
competitive binding
assay.
101881 Two antibodies may be determined to bind to the same epitope if
essentially all amino
acid mutations in the antigen that reduce or eliminate binding of one antibody
reduce or
eliminate binding of the other. Two antibodies may be determined to bind to
overlapping
epitopes if only a subset of the amino acid mutations that reduce or eliminate
binding of one
antibody reduce or eliminate binding of the other.
101891 In certain embodiments, the antibody (i) comprises an immunoglobulin
heavy chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 86%,
87%, 88%, 89%, 89.5%, 90%, 90.5%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99%
identical to SEQ ID NO: 164, and an immunoglobulin light chain variable region
comprising an
amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%,
92%, 93%, 94%, 94.5%, 95%, 95.5%, 96%, 97%, 98%, or 99% identical to SEQ ID
NO: 167,
and (ii) competes for binding to human PD-Li with and/or binds to same epi
tope on human PD-
L1 as an antibody comprising an immunoglobulin heavy chain variable region
comprising the
amino acid sequence of SEQ ID NO: 164, and an immunoglobulin light chain
variable region
comprising the amino acid sequence of SEQ ID NO: 167.
101901 In certain embodiments, the antibody (i) comprises an immunoglobulin
heavy chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 86%,
87%, 88%, 89%, 89.5%, 90%, 90.5%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99%
identical to SEQ ID NO: 199, and an immunoglobulin light chain variable region
comprising an
amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%,
92%, 93%, 94%, 94.5%, 95%, 95.5%, 96%, 97%, 98%, or 99% identical to SEQ ID
NO: 200,
and (ii) competes for binding to human PD-Li with and/or binds to same epitope
on human PD-
Li as an antibody comprising an immunoglobulin heavy chain variable region
comprising the
amino acid sequence of SEQ ID NO: 199, and an immunoglobulin light chain
variable region
comprising the amino acid sequence of SEQ ID NO: 200.
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[0191] In certain embodiments, the antibody (i) comprises an immunoglobulin
heavy chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 86%,
87%, 88%, 89%, 89.5%, 90%, 90.5%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99%
identical to SEQ ID NO: 199, and an immunoglobulin light chain variable region
comprising an
amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%,
92%, 93%, 94%, 94.5%, 95%, 95.5%, 96%, 97%, 98%, or 99%, and (ii) competes for
binding to
human PD-Li with and/or binds to same epitope on human PD-Li as an antibody
comprising an
immunoglobulin heavy chain variable region comprising the amino acid sequence
of SEQ ID
NO: 199, and an immunoglobulin light chain variable region comprising the
amino acid
sequence of SEQ ID NO: 201.
[0192] In certain embodiments, the antibody (i) comprises an immunoglobulin
heavy chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 86%,
87%, 88%, 89%, 89.5%, 90%, 90.5%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99%
identical to SEQ ID NO: 199, and an immunoglobulin light chain variable region
comprising an
amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%,
92%, 93%, 94%, 94.5%, 95%, 95.5%, 96%, 97%, 98%, or 99% identical to SEQ ID
NO: 202,
and (ii) competes for binding to human PD-Li with and/or binds to same epitope
on human PD-
Li as an antibody comprising an immunoglobulin heavy chain variable region
comprising the
amino acid sequence of SEQ ID NO: 199, and an immunoglobulin light chain
variable region
comprising the amino acid sequence of SEQ ID NO: 202.
[0193] In certain embodiments, the antibody (i) comprises an immunoglobulin
heavy chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 86%,
87%, 88%, 89%, 89.5%, 90%, 90.5%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99%
identical to SEQ ID NO: 199, and an immunoglobulin light chain variable region
comprising an
amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%,
92%, 93%, 94%, 94.5%, 95%, 95.5%, 96%, 97%, 98%, or 99% identical to SEQ ID
NO: 204,
and (ii) competes for binding to human PD-Li with and/or binds to same epitope
on human PD-
Li as an antibody comprising an immunoglobulin heavy chain variable region
comprising the
amino acid sequence of SEQ ID NO: 199, and an immunoglobulin light chain
variable region
comprising the amino acid sequence of SEQ ID NO: 204
101941 In certain embodiments, the antibody (i) comprises an immunoglobulin
heavy chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 132, and an immunoglobulin
light chain
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variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 136, and (ii) competes for
binding to
human PD-Li with and/or binds to same epitope on human PD-Li as an antibody
comprising an
immunoglobulin heavy chain variable region comprising the amino acid sequence
of SEQ ID
NO: 132, and an immunoglobulin light chain variable region comprising the
amino acid
sequence of SEQ ID NO: 136.
101951 In certain embodiments, the antibody (i) comprises an immunoglobulin
heavy chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 140, and an immunoglobulin
light chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 144, and (ii) competes for
binding to
human PD-Li with and/or binds to same epitope on human PD-Li as an antibody
comprising an
immunoglobulin heavy chain variable region comprising the amino acid sequence
of SEQ ID
NO: 140, and an immunoglobulin light chain variable region comprising the
amino acid
sequence of SEQ ID NO: 144.
101961 In certain embodiments, the antibody (i) comprises an immunoglobulin
heavy chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 148, and an immunoglobulin
light chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 152, and (ii) competes for
binding to
human PD-Li with and/or binds to same epitope on human PD-Li as an antibody
comprising an
immunoglobulin heavy chain variable region comprising the amino acid sequence
of SEQ ID
NO: 148, and an immunoglobulin light chain variable region comprising the
amino acid
sequence of SEQ ID NO: 152.
101971 In certain embodiments, the antibody (i) comprises an immunoglobulin
heavy chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 156, and an immunoglobulin
light chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 160, and (ii) competes for
binding to
human PD-Li with and/or binds to same epitope on human PD-Li as an antibody
comprising an
immunoglobulin heavy chain variable region comprising the amino acid sequence
of SEQ ID
NO: 156, and an immunoglobulin light chain variable region comprising the
amino acid
sequence of SEQ ID NO: 160.
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101981 In certain embodiments, the antibody (i) comprises an immunoglobulin
heavy chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 170, and an immunoglobulin
light chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 174, and (ii) competes for
binding to
human PD-Li with and/or binds to same epitope on human PD-Li as an antibody
comprising an
immunoglobulin heavy chain variable region comprising the amino acid sequence
of SEQ ID
NO: 170, and an immunoglobulin light chain variable region comprising the
amino acid
sequence of SEQ ID NO: 174.
101991 In certain embodiments, the antibody (i) comprises an immunoglobulin
heavy chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 178, and an immunoglobulin
light chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 182, and (ii) competes for
binding to
human PD-Li with and/or binds to same epitope on human PD-Li as an antibody
comprising an
immunoglobulin heavy chain variable region comprising the amino acid sequence
of SEQ ID
NO: 178, and an immunoglobulin light chain variable region comprising the
amino acid
sequence of SEQ ID NO: 182.
102001 In certain embodiments, the antibody (i) comprises an immunoglobulin
heavy chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 186, and an immunoglobulin
light chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ Ill NO: 190, and (ii) competes for
binding to
human PD-Li with and/or binds to same epitope on human PD-Li as an antibody
comprising an
immunoglobulin heavy chain variable region comprising the amino acid sequence
of SEQ ID
NO: 186, and an immunoglobulin light chain variable region comprising the
amino acid
sequence of SEQ ID NO: 190.
102011 In certain embodiments, the antibody (i) comprises an immunoglobulin
heavy chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 194, and an immunoglobulin
light chain
variable region comprising an amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 198, and (ii) competes for
binding to
human PD-Li with and/or binds to same epitope on human PD-Li as an antibody
comprising an
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immunoglobulin heavy chain variable region comprising the amino acid sequence
of SEQ ID
NO: 194, and an immunoglobulin light chain variable region comprising the
amino acid
sequence of SEQ ID NO: 198.
[0202] The antibodies disclosed herein may be further optimized (e.g.,
affinity-matured) to
improve biochemical characteristics including affinity and/or specificity,
improve biophysical
properties including aggregation, stability, precipitation and/or non-specific
interactions, and/or
to reduce immunogenicity. Affinity-maturation procedures are within ordinary
skill in the art.
For example, diversity can be introduced into an immunoglobulin heavy chain
and/or an
immunoglobulin light chain by DNA shuffling, chain shuffling, CDR shuffling,
random
mutagenesis and/or site-specific mutagenesis.
[0203] In certain embodiments, isolated human antibodies contain one or more
somatic
mutations. In these cases, antibodies can be modified to a human germline
sequence to optimize
the antibody (i.e., a process referred to as germlining).
102041 Generally, an optimized antibody has at least the same, or
substantially the same, affinity
for the antigen as the non-optimized (or parental) antibody from which it was
derived
Preferably, an optimized antibody has a higher affinity for the antigen when
compared to the
parental antibody.
[0205] If the antibody is for use as a therapeutic, it can be conjugated to an
effector agent such
as a small molecule toxin or a radionuclide using standard in vitro
conjugation chemistries. If
the effector agent is a polypeptide, the antibody can be chemically conjugated
to the effector or
joined to the effector as a fusion protein. Construction of fusion proteins is
within ordinary skill
in the art.
[0206] The antibody can be conjugated to an effector moiety such as a small
molecule toxin or a
radionuclide using standard in vitro conjugation chemistries. If the effector
moiety is a
polypeptide, the antibody can be chemically conjugated to the effector or
joined to the effector
as a fusion protein. Construction of fusion proteins is within ordinary skill
in the art.
II. Sialidase anti-PD-L1 Fusion Proteins
102071 To promote the selective removal of sialic acids on cells, e.g.,
hypersialylated cancer
cells such as PD-Li expressing cancer cells, and/or in the tumor
microenvironment, it may be
helpful to target a sialidase as described herein to such a cell or to such a
tumor
microenvironment. Additionally, in order to promote the removal of sialic acid
by a sialidase in
a subject, it may be helpful to extend the plasma half-life of the sialidase
in the subject. These
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can be achieved by including the sialidase in a fusion protein and/or antibody
conjugate (e.g., a
chemically conjugated conjugate).
102081 Accordingly, the invention further provides fusion proteins comprising
a sialidase
enzyme, or a functional fragment thereof, and a portion or fragment of an anti-
PD-Li antibody,
such as an immunoglobulin Fc domain (also referred to herein as an Fc domain),
or an
immunoglobulin antigen-binding domain (also referred to herein as an antigen-
binding domain).
In certain embodiments, the sialidase and anti-PD-Li antibody or portion
thereof (e.g.,
immunoglobulin Fc domain or antigen-binding domain) are linked by a peptide
bond or an
amino acid linker.
102091 As used herein, unless otherwise indicated, the term "fusion protein"
is understood to
refer to a single polypeptide chain comprising amino acid sequences based upon
two or more
separate proteins or polypeptide chains, where the two amino acid sequences
may be fused
together directly or via an intervening linker sequence, e.g., via an
intervening amino acid linker.
A nucleotide sequence encoding such a fusion protein can, for example, be
created using
conventional recombinant DNA technologies.
102101 In certain embodiments, a fusion protein comprises a tag, such as a
Strep tag (e.g., a
Strep II tag), a His tag (e.g., a 10x His tag), a myc tag, or a FLAG tag. The
tag can be located on
the C-terminus or the N-terminus of the fusion protein.
a. Sialidase Portion
102111 As used herein, the term "sialidase" refers to any enzyme, or a
functional fragment
thereof, that cleaves a terminal sialic acid residue from a substrate, for
example, a glycoprotein
or a glycolipid. The term sialidase includes variants having one or more amino
acid
substitutions, deletions, or insertions relative to a wild-type sialidase
sequence, and/or fusion
proteins or conjugates including a sialidase. Sialidases are also called
neuraminidases, and,
unless indicated otherwise, the two terms are used interchangeably herein. As
used herein, the
term "functional fragment" of a sialidase refers to fragment of a full-length
sialidase that retains,
for example, at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 95%, or 100% of the enzymatic
activity of the
corresponding full-length, naturally occurring sialidase. Sialidase enzymatic
activity may be
assayed by any method known in the art, including, for example, by measuring
the release of
sialic acid from the fluorogenic substrate 4-methylumbelliferyl-N-
acetylneuraminic acid (4MU-
NeuAc). In certain embodiments, the functional fragment comprises at least
100, 150, 200, 250,
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300, 310, 320, 330, 340, 350, 360, or 370 consecutive amino acids present in a
full-length,
naturally occurring sialidase.
102121 In certain embodiments, a sialidase portion of a sialidase-anti-PD-L1
fusion protein is
derived from a eukaryotic sialidase, e.g., a mammalian sialidase, e.g., a
human or mouse
sialidase.
102131 Four sialidases are encoded in the human genome: Neul, Neu2, Neu3 and
Neu4.
Human Neul is a lysosomal neuraminidase enzyme which functions in a complex
with beta-
galactosidase and cathepsin A. The amino acid sequence of human Neul is
depicted in SEQ ID
NO: 7, and a nucleotide sequence encoding human Neul is depicted in SEQ ID NO:
23.
102141 Human Neu2 is a cytosolic sialidase enzyme. The amino acid sequence of
human Neu2
is depicted in SEQ ID NO: 1, and a nucleotide sequence encoding human Neu2 is
depicted in
SEQ ID NO: 24. Unless stated otherwise, as used herein, wild-type human Neu2
refers to
human Neu2 having the amino acid sequence of SEQ ID NO: 1.
102151 Human Neu3 is a plasma membrane sialidase with an activity specific for
gangliosides.
Human Neu3 has two isoforms: isoform 1 and isoform 2. The amino acid sequence
of human
Neu3, isoform 1 is depicted in SEQ ID NO: 8, and a nucleotide sequence
encoding human Neu3,
isoform 1 is depicted in SEQ ID NO: 25. The amino acid sequence of human Neu3,
isoform 2 is
depicted in SEQ ID NO: 9, and a nucleotide sequence encoding human Neu3,
isoform 2 is
depicted in SEQ ID NO: 34.
102161 Human Neu4 has two isoforms: isoform 1 is a peripheral membrane protein
and isoform
2 localizes to the lysosome lumen. The amino acid sequence of human Neu4,
isoform 1 is
depicted in SEQ ID NO: 10, and a nucleotide sequence encoding human Neu4,
isoform 1 is
depicted in SEQ ID NO: 26. The amino acid sequence of human Neu4, isoform 2 is
depicted in
SEQ ID NO: 11, and a nucleotide sequence encoding human Neu4, isoform 2 is
depicted in SEQ
ID NO: 35.
102171 Four sialidases have also been found in the mouse genome and are
referred to as Neul,
Neu2, Neu3 and Neu4. The amino acid sequence of mouse Neul is depicted in SEQ
ID NO: 38,
and a nucleotide sequence encoding mouse Neul is depicted in SEQ ID NO: 42.
The amino acid
sequence of mouse Neu2 is depicted in SEQ ID NO: 39 and a nucleotide sequence
encoding
mouse Neu2 is depicted in SEQ ID NO: 43. The amino acid sequence of mouse Neu3
is
depicted in SEQ TD NO: 40, and a nucleotide sequence encoding mouse Neu3 is
depicted in
SEQ ID NO: 44. The amino acid sequence of mouse Neu4 is depicted in SEQ ID NO:
41, and a
nucleotide sequence encoding mouse Neu4 is depicted in SEQ ID NO: 45.
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102181 In certain embodiments, a sialidase portion of a sialidase-anti-PD-L1
fusion protein is
derived from a prokaryotic sialidase. Exemplary prokaryotic sialidases include
sialidases from
Salmonella typhimurium and Vibrio cholera. The amino acid sequence of
Salmonella
typhimurinm sialidase (St-sialidase) is depicted in SEQ ID NO: 30, and a
nucleotide sequence
encoding Salmonella typhimurium sialidase is depicted in SEQ ID NO: 6. The
amino acid
sequence of Vibrio cholera sialidase is depicted in SEQ ID NO: 36, and a
nucleotide sequence
encoding Vibrio cholera sialidase is depicted in SEQ ID NO: 37.
102191 In certain embodiments, the sialidase portion of a sialidase-anti-PD-L1
fusion protein is a
mutant sialidase, e.g., a recombinant mutant human sialidase. In certain
embodiments, the
recombinant mutant human sialidase has about 5%, about 10%, about 15%, about
20%, about
25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about
60%, about
65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about
100%, or
more than 100% of the enzymatic activity of a corresponding (or template) wild-
type human
sialidase.
[0220] In certain embodiments, the recombinant mutant human sialidase has the
same substrate
specificity as the corresponding wild-type human sialidase. In other
embodiments, the
recombinant mutant human sialidase has a different substrate specificity than
the corresponding
wild-type human sialidase. For example, in certain embodiments the recombinant
mutant human
sialidase can cleave a2,3, a2,6, and/or a2,8 linkages. In certain embodiments
the sialidase can
cleave a2,3 and a2,8 linkages.
[0221] In certain embodiments, the expression yield of the recombinant mutant
human sialidase
in mammalian cells, e.g., HEK293 cells, CHO cells, murine myeloma cells (NSO,
Sp2/0), or
human fibrosarcoma cells (HT-1080), e.g., HEK293 cells, is greater than about
10%, about 20%,
about 50%, about 75%, about 100%, about 150%, about 200%, about 250%, about
300%, about
400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about
1,000% of the
expression yield of the corresponding wild-type human sialidase.
[0222] In certain embodiments, the recombinant mutant human sialidase has
about 5%, about
10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about
50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about
85%, about
90%, about 95%, about 100%, or more than 100% of the enzymatic activity of a
corresponding
wild-type human sialidase, and the expression yield of the recombinant mutant
human sialidase
in mammalian cells, e.g., HEK293 cells, is greater than about 10%, about 20%,
about 50%,
about 75%, about 100%, about 150%, about 200%, about 250%, about 300%, about
400%, about
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500%, about 600%, about 700%, about 800%, about 900%, or about 1,000% of the
expression
yield of a corresponding wild-type human sialidase.
102231 In certain embodiments, the amino acid sequence of the recombinant
mutant human
sialidase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%,
or 99% sequence identity to the amino acid sequence of a corresponding wild-
type human
sialidase.
1. Substitution of Cysteine Residues
102241 In certain embodiments, the recombinant mutant human sialidase
comprises a
substitution of at least one cysteine (cys, C) residue. It has been discovered
that certain cysteine
residues in sialidases may inhibit expression of functional protein as a
result of protein
aggregation. Accordingly, in certain embodiments, the recombinant mutant human
sialidase
contains at least one mutation to remove a free cysteine (e.g., for Neul (SEQ
ID NO: 7), a
mutation of, for example, one or more of C111, C117, C171, C183, C218, C240,
C242, and
C252; for Neu2 (SEQ ID NO: 1), a mutation of, for example, one or more of
C125, C196, C219,
C272, C332, and C352; for Neu3 (SEQ ID NO: 8), a mutation of, for example, one
or more of
C7, C90, C99, C106, C127, C136, C189, C194, C226, C242, C250, C273, C279,
C295, C356,
C365, C368, C384, C383, C394, and C415, and for Neu4 (SEQ ID NO: 10), a
mutation of, for
example, one or more of C88, C125, C126, C186, C191, C211, C223, C239, C276,
C437, C453,
C480, and C481). Free cysteines can be substituted with any amino acid. In
certain
embodiments, the free cysteine is substituted with serine (ser, S), isoleucine
(iso, I), valine (val,
V), phenylalanine (phe, F), leucine (leu, L), or alanine (ala, A). Exemplary
cysteine
substitutions in Neu2 include C125A, C1251, C125S, C125V, C196A, C196L, C196V,
C2725,
C272V, C332A, C332S, C332V, C352L, and C352V.
102251 In certain embodiments, the recombinant mutant human sialidase
comprises two or more
cysteine substitutions. Exemplary double or triple cysteine substitutions in
Neu2 include: C125S
and C332S; C272V and C332A; C272V and C332S; C332A and C352L; C125S and C196L;
C196L and C352L; C196L and C332A; C332A and C352L; and C196L, C332A and C352L.
102261 In certain embodiments, the recombinant mutant human sialidase is a
Neu2 sialidase and
comprises the substitutions C322A and C352L.
102271 In certain embodiments, the sialidase contains an amino acid
substitution at 2, 3, 4, 5, or
6 cysteines typically present in a human sialidase, e.g., Neu2 or Neu3.
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102281 In certain embodiments, the recombinant mutant human sialidase
comprises a
substitution or combination of substitutions corresponding to a substitution
or combination of
substitutions listed in TABLE 1 (amino acid positions corresponding to wild-
type human Neu2
(SEQ ID NO: 1)).
TABLE 1
Substitution(s)
C125A
C1251
C125S
C125V
C196A
C196L
C196V
C272S
C272V
C332A
C332S
C332V
C352L
C352V
C1255 + C3325
C272V + C332A
C272V + C332S
C332A + C352L
C125S +C196L
C196L + C352L
C196L + C332A
C196L + C332A + C352L
2. Substitutions of Residues to Increase pI and/or Decrease Hydrophobicity
102291 The isoelectric point (pI) of a protein is the pH at which the net
charge is zero. The pI
also generally indicates the pH at which the protein is least soluble, which
may affect the ability
to express and purify the protein. Generally, a protein has good solubility if
its pI is greater than
2 units above the pH of the solution. Human Neu2 has a predicted pI of 7.5.
Thus, human Neu2
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is least soluble around neutral pH, which is undesirable because expression
and physiological
systems are at neutral pH. In contrast, the sialidase from Salmonella
typhimurium (St-sialidase),
which exhibits good solubility and recombinant expression, has a pI of 9.6.
Accordingly, to
increase expression of human Neu2 or the other human sialidases, a recombinant
mutant human
sialidase may be designed to contain one or more amino acid substitution(s)
wherein the
substitution(s) increase(s) the pI of the sialidase relative to a sialidase
without the substitution.
Additionally, decreasing the number of hydrophobic amino acids on the surface
of a sialidase
may improve expression of sialidase by, for example, reducing aggregation.
Accordingly, to
increase expression of human Neu2 or the other human sialidases, a recombinant
mutant human
sialidase may be designed to contain one or more amino acid substitution(s)
wherein the
substitution(s) decrease(s) the hydrophobicity of a surface of the sialidase
relative to a sialidase
without the substitution(s).
102301 Accordingly, in certain embodiments, the recombinant mutant human
sialidase comprises
at least one amino acid substitution, wherein the substitution increases the
isoelectric point (pI)
of the sialidase and/or decreases the hydrophobicity of the sialidase relative
to a sialidase
without the substitution. This may be achieved by introducing one or more
charged amino acids,
for example, positively or negatively charged amino acids, into the
recombinant sialidase. In
certain embodiments, the amino acid substitution is to a charged amino acid,
for example, a
positively charged amino acid such as lysine (lys, K), histidine (his, H), or
arginine (arg, R), or a
negatively charged amino acid such as aspartic acid (asp, D) or glutamic acid
(glu, E). In certain
embodiments, the amino acid substitution is to a lysine residue. In certain
embodiments, the
substitution increases the pI of the sialidase to about 7.75, about 8, about
8.25, about 8.5, about
8.75, about 9, about 9.25, about 9.5, or about 9.75.
102311 In certain embodiments, the amino acid substitution occurs at a surface
exposed D or E
amino acid, in a helix or loop, or in a position that has a K or R in the
corresponding position of
St-sialidase. In certain embodiments, the amino acid substitution occurs at an
amino acid that is
remote from the catalytic site or otherwise not involved in catalysis, an
amino acid that is not
conserved with the other human Neu proteins or with St-Sialidase or
Clostridium NanH, or an
amino acid that is not located in a domain important for function (e.g., an
Asp-box or beta
strand)
102321 Exemplary amino acid substitutions in Neu2 that increase the
isoelectric point (pI) of the
sialidase and/or decrease the hydrophobicity of the sialidase relative to a
sialidase without the
substitution include A2E, A2K, D215K, V325E, V325K, E257K, and E319K. In
certain
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embodiments, the recombinant mutant human sialidase comprises two or more
amino acid
substitutions, including, for example, A2K and V325E, A2K and V325K, E257K and
V325K,
A2K and E257K, and E257K and A2K and V325K.
[0233] In certain embodiments, the recombinant mutant human sialidase
comprises a
substitution or combination of substitutions corresponding to a substitution
or combination of
substitutions listed in TABLE 2 (amino acid positions corresponding to wild-
type human Neu2
(SEQ NO: 1)).
TABLE 2
Substitution(s)
A2K
E72K
D215K
E257K
V325K
A2K + E257K
A2K + V325E
A2K + V325K
E257K + V325K
3. Addition of N-terminal Peptides and N- or C-terminal Substitutions
[0234] It has been discovered that the addition of a peptide sequence of two
or more amino acids
to the N-terminus of a human sialidase can improve expression and/or activity
of the sialidase.
In certain embodiments, the peptide is at least 2 amino acids in length, for
example, from 2 to
20, from 2 to 10, from 2 to 5, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20
amino acids in length. In certain embodiments, the peptide may form, or have a
propensity to
form, an a-helix.
[0235] In mice, a Neu2 isoform (type B) found in thymus contains six amino
acids not present in
the canonical isoform of Neu2 found in skeletal muscle. In certain embodiments
herein, the N-
terminal six amino acids of the mouse thymus Neu2 isoform, MEDLRP (SEQ ID NO:
4), or
variations thereof, can be added onto a human Neu, e.g., human Neu2. In
certain embodiments,
the recombinant mutant human sialidase comprises a peptide at least two amino
acid residues in
length covalently associated with an N-terminal amino acid of the sialidase.
In certain
embodiments the recombinant mutant human sialidase comprises the peptide
MEDLRP (SEQ ID
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NO: 4) or EDLRP (SEQ ID NO: 3) covalently associated with an N-terminal amino
acid of the
sialidase. In certain embodiments, the sialidase may further comprise a
cleavage site, e.g., a
proteolytic cleavage site, located between the peptide, e.g., MEDLRP (SEQ ID
NO: 4) or
EDLRP (SEQ ID NO: 3), and the remainder of the sialidase. In certain
embodiments, the
peptide, e.g., MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3), may be post-
translationally cleaved from the remainder of the sialidase.
[0236] Alternatively to, or in combination with, the N-terminal addition, 1-5
amino acids of the
12 amino acid N-terminal region of the recombinant mutant human sialidase may
be removed,
e.g., the N-terminal methionine can be removed. In certain embodiments, if the
recombinant
mutant human sialidase is Neu2, the N-terminal methionine can be removed, the
first five amino
acids (MASLP; SEQ ID NO: 12) can be removed, or the second through fourth
amino acids
(ASLP; SEQ ID NO: 13) can be removed.
[0237] In certain embodiments, 1-5 amino acids of the 12 amino acid N-terminal
region of the
recombinant mutant human sialidase are substituted with MEDLRP (SEQ ID NO: 4),
EDLRP
(SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14). For example, in certain
embodiments, if
the recombinant mutant human sialidase is Neu2, the amino acids MASLP (SEQ ID
NO: 12),
ASLP (SEQ ID NO: 13) or M are substituted with MEDLRP (SEQ ID NO: 4), EDLRP
(SEQ ID
NO: 3) or TVEKSVVF (SEQ ID NO: 14).
[0238] Human sialidases have a 3-propeller structure, characterized by 6 blade-
shaped 13-sheets
arranged toroidally around a central axis. Generally, hydrophobic interactions
between the
blades of a 13-propeller, including between the N- and C-terminal blades,
enhance stability.
Accordingly, in order to increase expression of human Neu2 or the other human
sialidases, a
recombinant mutant human sialidase can be designed comprising an amino acid
substitution that
increases hydrophobic interactions and/or hydrogen bonding between the N- and
C-terminal 13-
propeller blades of the sialidase.
[0239] Accordingly, in certain embodiments, the recombinant mutant human
sialidase comprises
a substitution of at least one wild-type amino acid residue, wherein the
substitution increases
hydrophobic interactions and/or hydrogen bonding between the N- and C-termini
of the sialidase
relative to a sialidase without the substitution. In certain embodiments, the
wild-type amino acid
is substituted with asparagine (asn, N), lysine (lys, K), tyrosine (tyr, Y),
phenylalanine (phe, F),
or tryptophan (trp, W). Exemplary substitutions in Neu2 that increase
hydrophobic interactions
and/or hydrogen bonding between the N- and C-termini include L4N, L4K, V6Y,
L7N, L4N and
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L7N, L4N and V6Y and L7N, V12N, V12Y, V12L, V6Y, V6F, or V6W. In certain
embodiments, the sialidase comprises the V6Y substitution.
102401 In certain embodiments, the recombinant mutant human sialidase
comprises a
combination of the above substitutions. For example, a recombinant mutant
human Neu2
sialidase can comprise the additional amino acids MEDLRP (SEQ ID NO: 4), EDLRP
(SEQ ID
NO: 3), or TVEKSVVF (SEQ ID NO: 14) at the N-terminus and, in combination, can
comprise
at least one L4N, L4K, V6Y, L7N, L4N and L7N, L4N and V6Y and L7N, V12N, V12Y,
V12L,
V6Y, V6F, or V6W substitution. In certain embodiments, the amino acids MASLP
(SEQ ID
NO: 12), ASLP (SEQ ID NO: 13) or M of a recombinant mutant human Neu2
sialidase are
replaced with MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3) or TVEKSVVF (SEQ ID
NO: 14) and the recombinant mutant human Neu2 sialidase also comprises at
least one L4N,
L4K, V6Y, L7N, L4N and L7N, L4N and V6Y and L7N, V12N, V12Y, V12L, V6Y, V6F,
or
V6W substitution.
102411 In certain embodiments, the recombinant mutant human sialidase
comprises a mutation
or combination of mutations corresponding to a mutation or combination of
mutations listed in
TABLE 3 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID
NO: 1)).
TABLE 3
Mutation(s)
Substitute M at the N-terminus with EDLRP (SEQ ID NO: 3)
Substitute M at the N-terminus with MEDLRP (SEQ 1D NO: 4)
Insert MEDLRP (SEQ ID NO: 4) at the N-terminus
Substitute MASLP (SEQ ID NO: 12) at the N-terminus with
MEDLRP (SEQ ID NO: 4)
L4N
V6Y
L7N
V6F
V6W
102421 Additionally, in certain embodiments, the sialidase comprises a
substitution or deletion of
an N-terminal methionine at the N-terminus of the sialidase. For example, in
certain
embodiments, the sialidase comprises a substitution of a methionine residue at
a position
corresponding to position 1 of wild-type human Neu2 (SEQ ID NO: 1), e.g., the
methionine at a
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position corresponding to position 1 of wild-type human Neu2 is substituted by
alanine (MIA)
or aspartic acid (M1D). In other embodiments, the sialidase comprises a
deletion of a
methionine residue at a position corresponding to position 1 (AMI) of wild-
type human Neu2
(SEQ ID NO: 1).
102431 In certain embodiments, the recombinant mutant human sialidase
comprises a
substitution or combination of substitutions corresponding to a substitution
or combination of
substitutions listed in TABLE 4 (amino acid positions corresponding to wild-
type human Neu2
(SEQ ID NO: 1)).
TABLE 4
Mutation(s)
Deletion of Ml, V6Y, I187K
M1 R, V6Y, I187K
M1H, V6Y, I187K
M1K, V6Y, I187K
MID, V6Y, II87K
MIT, V6Y, I187K
M1N, V6Y, I187K
M1Q, V6Y, I187K
M1G, V6Y, I187K
M1A, V6Y, I187K
M1V, V6Y, I187K
V6Y, I187K
M1F, V6Y, I187K
MlY, V6Y, I187K
4. Substitutions of Residues to Decrease Proteolytic Cleavage
102441 It has been discovered that certain sialidases (e.g., human Neu2) are
susceptible to
cleavage by a protease (e.g., trypsin). As a result, proteolytic cleavage of
the sialidase may
occur during recombinant protein production, harvesting, purification, or
formulation, during
administration to a subject, or after administration to a subject.
Accordingly, in certain
embodiments, the recombinant mutant human sialidase comprises a substitution
of at least one
wild-type amino acid residue, wherein the substitution decreases cleavage of
the sialidase by a
protease (e.g., trypsin) relative to a sialidase without the substitution.
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102451 In certain embodiments, incubation of the recombinant mutant human
sialidase with a
protease (e.g., trypsin) results in from about 1% to about 50%, from about 1%
to about 40%,
from about 1%, to about 30%, from about 1% to about 20%, from about 1% to
about 10%, from
about 1% to about 5%, from about 5% to about 50%, from about 5% to about 40%,
from about
5% to about 30%, from about 5% to about 20%, from about 5% to about 10%, from
about 10%
to about 50%, from about 10% to about 40%, from about 10% to about 30%, from
about 10% to
about 20%, from about 20% to about 50%, from about 20% to about 40%, from
about 20% to
about 30%, from about 30% to about 50%, from about 30% to about 40%, or from
about 40% to
about 50% of the proteolytic cleavage of a corresponding wild-type sialidase
when incubated
with the protease under the same conditions. In certain embodiments,
incubation of the
recombinant mutant human sialidase with a protease (e.g., trypsin) results in
less than 50%, less
than 40%, less than 30%, less than 10%, less than 5%, less than 3%, less than
1%, or less than
0.5% of the proteolytic cleavage of a corresponding wild-type sialidase when
incubated with the
protease under the same conditions. Proteolytic cleavage can be assayed by any
method known
in the art, including for example, by SDS-PAGE as described in Example 4
herein.
102461 Exemplary substitutions that increase resistance to proteolytic
cleavage include: (i) a
substitution of an alanine residue at a position corresponding to position 242
of wild-type human
Neu2 (SEQ ID NO. 1), e.g., a substitution by cysteine (A242C), phenylalanine
(A242F), glycine
(A242G), histidine (A242H), isoleucine (A242I), lysine (A242K), leucine
(A242L), methionine
(A242M), asparagine (A242N), glutamine (A242Q), arginine (A242R), serine
(A242S), valine
(A242V), tryptophan (A242W), or tyrosine (A242Y); (ii) a substitution of an
arginine residue at
a position corresponding to position 243 of wild-type human Neu2 (SEQ ID NO:
1), e.g., a
substitution by glutamic acid (R243E), histidine (R243H), asparagine (R243N),
glutamine
(R243Q), or lysine (R243K); (iii) a substitution of a valine residue at a
position corresponding to
position 244 of wild-type human Neu2 (SEQ ID NO: 1), e.g., a substitution by
isoleucine
(V244I), lysine (V244K), or proline (V244P); or (iv) a combination of any of
the foregoing. In
certain embodiments, the recombinant mutant human sialidase comprises a
substitution selected
from A242C, A242F, A242Y, and A242W. In certain embodiments, the recombinant
mutant
human sialidase comprises a substitution or a combination of substitutions
corresponding to a
substitution or combination of substitutions listed in TABLE 5 (amino acid
positions
corresponding to wild-type human Neu2 (SEQ ID NO: 1)).
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TABLE 5
Wild Type Exemplary Substitution(s) at Specified
Position(s)
Human Neu2
(SEQ ID NO: 1)
Amino Acid
A242 C, F, G, H, I, K, L, M, N, P. Q, R, S, V, W, Y
R243 E, H, N, Q, K
V244 I, K, P
102471 Additional exemplary substitutions that increase resistance to
proteolytic cleavage
(and/or increase expression yield and/or enzymatic activity) include: (i) a
substitution of a
leucine residue at a position corresponding to position 240 of wild-type human
Neu2 (SEQ ID
NO: 1), e.g., a substitution by aspartic acid (L240D), asparagine (L240N), or
tyrosine (L240Y);
(ii) a substitution of an alanine residue at a position corresponding to
position 213 of wild-type
human Neu2 (SEQ ID NO: 1), e.g., a substitution by cysteine (A213C),
asparagine (A213N),
serine (A213S), or threonine (A213T); (iii) a substitution of an arginine
residue at a position
corresponding to position 241 of wild-type human Neu2 (SEQ ID NO: 1), e.g., a
substitution by
alanine (R241A), aspartic acid (R241D), leucine (R241L), glutamine (R241Q), or
tyrosine
(R241Y); (iv) a substitution of a serine residue at a position corresponding
to position 258 of
wild-type human Neu2 (SEQ ID NO: 1), e.g., a substitution by cysteine (S258C);
(v) a
substitution of a leucine residue at a position corresponding to position 260
of wild-type human
Neu2 (SEQ ID NO: 1), e.g., a substitution by aspartic acid (L260D),
phenylalanine (L260F),
glutamine (L260Q), or threonine (L260T); (vi) a substitution of a valine
residue at a position
corresponding to position 265 of wild-type human Neu2 (SEQ ID NO: 1), e.g., a
substitution by
phenylalanine (V265F); or (vii) a combination of any of the foregoing. It is
contemplated that,
in certain embodiments, a substitution or a combination of substitutions at
these positions may
improve hydrophobic and/or aromatic interaction between secondary structure
elements in the
sialidase (e.g., between an a-helix and the nearest 13-sheet) thereby
stabilizing the structure and
improving resistance to proteolytic cleavage.
102481 In certain embodiments, the recombinant mutant sialidase comprises a
mutation at
position L240. In certain embodiments, the recombinant mutant sialidase
comprises a
combination of mutations at positions (i) A213 and A242, (ii) A213, A242, and
S258, (iii) L240
and L260, (iv) R241 and A242, (v) A242 and L260, (vi) A242 and V265, or (vii)
L240 and
A242. In certain embodiments, the recombinant mutant human sialidase comprises
a
combination of substitutions selected from (i) A213C, A242F, and S258C, (ii)
A213C and
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A242F, (iii) A213T and A242F, (iv) R241Y and A242F, and (v) L240Y and A242F.
In certain
embodiments, the recombinant mutant human sialidase comprises a substitution
or combination
of substitutions corresponding to a substitution or combination of
substitutions listed in TABLE
6 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)).
TABLE 6
Substitution(s)
A242C, V244P
A242R, V244R
A242R, V244H
A242Y, V244P
A242T, V244P
A242N, V244P
A213 C, A242F
A213S, A242F
A213T, A242F
A213N, A242F
A213C, A242F, 5258C
A242F, L260F
A242F, V265F
L240Y
L240Y, L260F
L240D, L260T
L240N, L260T
L240N, L260D
L240N, L260Q
L240Y, A242F
R241A, A242F
R241Y, A242F
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5. Other Substitutions
102491 In certain embodiments, the recombinant mutant human sialidase
comprises at least one
of the following substitutions: I187K, A328E, K370N, or H210N. In certain
embodiments, a
recombinant mutant human Neu2 comprises the substitution of the amino acids
GDYDAPTHQVQW (SEQ ID NO: 15) with the amino acids S1VIDQGSTW (SEQ ID NO: 16)
or
STDGGKTW (SEQ ID NO: 17). In certain embodiments, a recombinant mutant human
Neu2
comprises the substitution of the amino acids PRPPAPEA (SEQ ID NO: 18) with
the amino
acids QTPLEAAC (SEQ ID NO: 19). In certain embodiments, a recombinant mutant
human
Neu2 comprises the substitution of the amino acids NPRPPAPEA (SEQ ID NO: 20)
with the
amino acids SQNDGES (SEQ ID NO: 21).
102501 In certain embodiments, the recombinant mutant human sialidase
comprises at least one
substitution at a position corresponding to V212, A213, Q214, D215, T216,
L217, E218, C219,
Q220, V221, A222, E223, V224, E225, or T225.
102511 In certain embodiments, the recombinant mutant human sialidase
comprises an amino
acid substitution at a position identified in TABLE 7 (amino acid positions
corresponding to
wild-type human Neu2 (SEQ ID NO: 1). In certain embodiments, the sialidase
comprises an
amino acid substitution identified in TABLE 7. In certain embodiments, the
sialidase comprises
a combination of any amino acid substitutions identified in TABLE 7.
TABLE 7
Wild Type Exemplary Substitution(s) at Specified
Position(s)
Human Neu2
(SEQ ID NO: 1)
Amino Acid
M1
L4 S, T, Y, L, F, A, P, V, I, N, D, or H
P5 G
V6
L7 F, Y, S, I, T, or N
K9
V12 L, A, P, V, N, D, or H
F13 S, N, R, K, T, G, D, E, or A
122 S, N, R, K, T, G, D, E, A, Y, L, F, P, V, I, or
H
A24 S, N, R, K, T, G, D, E, A, Y, L, F, P, V, I, or
H
L34 S, T, Y, L, F, A, P, V, I, N, D, or H
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Wild Type Exemplary Substitution(s) at Specified
Position(s)
Human Neu2
(SEQ ID NO: I)
Amino Acid
A36 S, T, Y, L, F, A, P, V, I, N, D, or H
A42 R or D
K44 R or E
1(45 A, E, or R
L54
P62 H, G, N, T, S, F, I, D, or E
H64 F, Y, S, I, T, or N
Q69
R78
D80
P89 S, T, Y, L, F, A, P, V, I, N, D, H, or M
A93 E or K
G107
Q108
Q112 R or K
C125 Y, F, or L
Q126 E, F, H, I, L, or Y
A150 V
T156 R, N, D, C, G, H, I, L, F, S. Y, V. A, P. or T
F157 R, N, D, C, G, H, I, L, F, S. Y, V, A, or P
A158 R, N, D, C, G, H, I, L, F, S, Y, V, A, P, or T
V159 R, N, D, C, G, H, I, L, F, S, Y, V, A, or P
G160 R, N, D, C, G, H, I, L, F, S, Y, V, A, P, or T
P161 R, N, D, C, G, H, I, L, F, S. Y, V, A, or P
G162 R, N, D, C, G, H, I, L, F, S, Y, V, A, P, or T
H163 R, N, D, C, G, H, I, L, F, S, Y, V, A, or P
C164 R, N, D, C, G, H, I, L, F, S, Y, V, A, P, or T
L165 R, N, D, C, G, H, I, L, F, S. Y, V, A, or P
R170
A171
V176 R, N, D, C, G, H, I, L, F, S, Y, V, P, or A
P177 S, T, Y, L, F, A, P, V, I, N, D, or 1-1
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Wild Type Exemplary Substitution(s) at Specified
Position(s)
Human Neu2
(SEQ ID NO: I)
Amino Acid
A178 S, T, Y, L, F, A, P, V, I, N, D, or H
L184 S. N, R, K, T, G, D, E, A, F, H, I, L, P. V. or
Y
H185 S, N, R, K, T, G, D, E, or A
P186 S, N, R, K, T, G, D, E, A, F, H, I, L, P. V. or
Y
1187 S, N, R, K, T, G, D, E, or A
Q188 P, S, N, R, K, T, G, D, E, or A
R189
P190 F, M, A. D, G, H, N, P. R, S. or T
1191 M, A, D, F, H, I, L, N, P, S, T, V. Y, E, G, K,
or R
A194 S, T, Y, L, F, A, P. V. I, N, D, or H
A213 C, N, S, or T
L217 R, N, D, C, G, H, I, L, F, S, Y, or V
C219 R, N, D, C, G, H, I, L, F, S, Y, or V
A222
E225
H239
L240 D, N, or Y
R241 A, D, L, Q, or Y
A242 C, F, G, H, I, K, L, M, N, Q, R, S, V, W, or Y
V244 I or P
T249 A
D251
E257
S258
L260 D, F, Q, or T
V265
Q270 S, T, A, H, P, or F
G271 S, N, R, K, T, G, D, E, or A
C272 S. N, R, K, T, G, D, E, A, C, H, Y, F, H, L, P.
or V
W292
S301 A, D, E, F, G, H, I, K, L, M, N, P, Q, T, V, W,
Y, C, or R
W302 A, D, E, F, G, H, I, L, M, N, P. Q, R, S, T, V.
Y, or K
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Wild Type Exemplary Substitution(s) at Specified
Position(s)
Human Neu2
(SEQ ID NO: I)
Amino Acid
E319
V325 F, Y, S, I, T, N, A, D, H, L, P, or V
L326 F, Y, S, I, T, N, A, D, H, L, P, or V
L327 F, Y, S, I, T, N, A, D, H, L, P, or V
C332 A, D, G, H, N, P, R, S, or T
Y359 A or S
V363 R, S, T, Y, L, F, A, P, V, I, N, D, or H
L365 K, Q, F, Y, S. I, T, N, A, D, H, L, P, or V
102521 For example, in certain embodiments, the recombinant mutant human
sialidase
comprises (a) a substitution of a proline residue at a position corresponding
to position 5 of
wild-type human Neu2 (P5); (b) a substitution of a lysine residue at a
position corresponding to
position 9 of wild-type human Neu2 (K9); (c) a substitution of an alanine
residue at a position
corresponding to position 42 of wild-type human Neu2 (A42); (d) a substitution
of a lysine
residue at a position corresponding to position 44 of wild-type human Neu2
(K44), (e) a
substitution of a lysine residue at a position corresponding to position 45 of
wild-type human
Neu2 (K45), (f) a substitution of a leucine residue at a position
corresponding to position 54 of
wild-type human Neu2 (L54); (g) a substitution of a proline residue at a
position corresponding
to position 62 of wild-type human Neu2 (P62), (h) a substitution of a
glutamine residue at a
position corresponding to position 69 of wild-type human Neu2 (Q69), (i) a
substitution of an
arginine residue at a position corresponding to position 78 of wild-type human
Neu2 (R78), (j) a
substitution of an aspartic acid residue at a position corresponding to
position 80 of wild-type
human Neu2 (D80), (k) a substitution of an alanine residue at a position
corresponding to
position 93 of wild-type human Neu2 (A93), (1) a substitution of a glycine
residue at a position
corresponding to position 107 of wild-type human Neu2 (G107); (m) a
substitution of a
glutamine residue at a position corresponding to position 108 of wild-type
human Neu2 (Q108);
(n) a substitution of a glutamine residue at a position corresponding to
position 112 of wild-type
human Neu2 (Q112); (o) a substitution of a cysteine residue at a position
corresponding to
position 125 of wild-type human Neu2 (C125); (p) a substitution of a glutamine
residue at a
position corresponding to position 126 of wild-type human Neu2 (Q126); (q) a
substitution of an
alanine residue at a position corresponding to position 150 of wild-type human
Neu2 (A150), (r)
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a substitution of a cysteine residue at a position corresponding to position
164 of wild-type
human Neu2 (C164); (s) a substitution of an arginine residue at a position
corresponding to
position 170 of wild-type human Neu2 (R170); (t) a substitution of an alanine
residue at a
position corresponding to position 171 of wild-type human Neu2 (A171); (u) a
substitution of a
glutamine residue at a position corresponding to position 188 of wild-type
human Neu2 (Q188);
(v) a substitution of an arginine residue at a position corresponding to
position 189 of wild-type
human Neu2 (R189); (w) a substitution of an alanine residue at a position
corresponding to
position 213 of wild-type human Neu2 (A213); (x) a substitution of a leucine
residue at a
position corresponding to position 217 of wild-type human Neu2 (L217); (y) a
substitution of a
glutamic acid residue at a position corresponding to position 225 of wild-type
human Neu2
(E225); (z) a substitution of a histidine residue at a position corresponding
to position 239 of
wild-type human Neu2 (H239); (aa) a substitution of a leucine residue at a
position
corresponding to position 240 of wild-type human Neu2 (L240); (bb) a
substitution of an
arginine residue at a position corresponding to position 241 of wild-type
human Neu2 (R241);
(cc) a substitution of an alanine residue at a position corresponding to
position 242 of wild-type
human Neu2 (A242); (dd) a substitution of a valine residue at a position
corresponding to
position 244 of wild-type human Neu2 (V244); (cc) a substitution of a
threonine residue at a
position corresponding to position 249 of wild-type human Neu2 (1249); (ff) a
substitution of an
aspartic acid residue at a position corresponding to position 251 of wild-type
human Neu2
(D251); (gg) a substitution of a glutamic acid residue at a position
corresponding to position 257
of wild-type human Neu2 (E257); (hh) a substitution of a serine residue at a
position
corresponding to position 258 of wild-type human Neu2 (S258); (ii) a
substitution of a leucine
residue at a position corresponding to position 260 of wild-type human Neu2
(L260); (jj) a
substitution of a valine residue at a position corresponding to position 265
of wild-type human
Neu2 (V265); (kk) a substitution of a glutamine residue at a position
corresponding to position
270 of wild-type human Neu2 (Q270); (11) a substitution of a tryptophan
residue at a position
corresponding to position 292 of wild-type human Neu2 (W292); (mm) a
substitution of a serine
residue at a position corresponding to position 301 of wild-type human Neu2
(S301); (nn) a
substitution of a tryptophan residue at a position corresponding to position
302 of wild-type
human Neu2 (W302); (oo) a substitution of a valine residue at a position
corresponding to
position 363 of wild-type human Neu2 (V363); or (pp) a substitution of a
leucine residue at a
position corresponding to position 365 of wild-type human Neu2 (L365); or a
combination of
any of the foregoing substitutions. For example, the sialidase may comprise a
substitution of
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K9, A42, P62, A93, Q216, A242, Q270, S301, W302, V363, or L365, or a
combination of any
of the foregoing substitutions.
102531 In certain embodiments, in the sialidase. (a) the proline residue at a
position
corresponding to position 5 of wild-type human Neu2 is substituted by
histidine (P5H); (b) the
lysine residue at a position corresponding to position 9 of wild-type human
Neu2 is substituted
by aspartic acid (K9D); (c) the alanine residue at a position corresponding to
position 42 of wild-
type human Neu2 is substituted by arginine (A42R) or aspartic acid (A42D); (d)
the lysine
residue at a position corresponding to position 44 of wild-type human Neu2 is
substituted by
arginine (K44R) or glutamic acid (K44E); (e) the lysine residue at a position
corresponding to
position 45 of wild-type human Neu2 is substituted by alanine (K45A), arginine
(K45R), or
glutamic acid (K45E), (f) the leucine residue at a position corresponding to
position 54 of wild-
type human Neu2 is substituted by methionine (L54M), (g) the proline residue
at a position
corresponding to position 62 of wild-type human Neu2 is substituted by
asparagine (P62N),
aspartic acid (P62D), histidine (P62H), glutamic acid (P62E), glycine (P62G),
serine (P62S), or
threonine (P62T), (h) the glutamine residue at a position corresponding to
position 69 of wild-
type human Neu2 is substituted by histidine (Q69H), (i) the arginine residue
at a position
corresponding to position 78 of wild-type human Neu2 is substituted by lysine
(R78K), (j) the
aspartic acid residue at a position corresponding to position 80 of wild-type
human Neu2 is
substituted by proline (D8OP); (k) the alanine residue at a position
corresponding to position 93
of wild-type human Neu2 is substituted by glutamic acid (A93E) or lysine
(A93K); (1) the
glycine residue at a position corresponding to position 107 of wild-type human
Neu2 is
substituted by aspartic acid (G1 07D); (m) the glutamine residue at a position
corresponding to
position 108 of wild-type human Neu2 is substituted by histidine (Q108H); (n)
the glutamine
residue at a position corresponding to position 112 of wild-type human Neu2 is
substituted by
arginine (Q1 12R) or lysine (Q1 12K); (o) the cysteine residue at a position
corresponding to
position 125 of wild-type human Neu2 is substituted by leucine (C125L); (p)
the glutamine
residue at a position corresponding to position 126 of wild-type human Neu2 is
substituted by
leucine (Q126L), glutamic acid (Q126E), phenylalanine (Q126F), histidine
(Q126H), isoleucine
(Q1261), or tyrosine (Q126Y); (q) the alanine residue at a position
corresponding to position 150
of wild-type human Neu2 is substituted by valine (A150V); (r) the cysteine
residue at a position
corresponding to position 164 of wild-type human Neu2 is substituted by
glycine (C164G); (s)
the arginine residue at a position corresponding to position 170 of wild-type
human Neu2 is
substituted by proline (R170P); (t) the alanine residue at a position
corresponding to position 171
of wild-type human Neu2 is substituted by glycine (A171G), (u) the glutamine
residue at a
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position corresponding to position 188 of wild-type human Neu2 is substituted
by proline
(Q188P); (v) the arginine residue at a position corresponding to position 189
of wild-type human
Neu2 is substituted by proline (R189P); (w) the alanine residue at a position
corresponding to
position 213 of wild-type human Neu2 is substituted by cysteine (A213C),
asparagine (A213N),
serine (A213S), or threonine (A213T); (x) the leucine residue at a position
corresponding to
position 217 of wild-type human Neu2 is substituted by alanine (L217A) or
valine (L217V); (y)
the threonine residue at a position corresponding to position 249 of wild-type
human Neu2 is
substituted by alanine (T249A); (z) the aspartic acid residue at a position
corresponding to
position 251 of wild-type human Neu2 is substituted by glycine (D251G); (aa)
the glutamic acid
residue at a position corresponding to position 225 of wild-type human Neu2 is
substituted by
proline (E225P); (bb) the histidine residue at a position corresponding to
position 239 of wild-
type human Neu2 is substituted by proline (H239P); (cc) the leucine residue at
a position
corresponding to position 240 of wild-type human Neu2 is substituted by
aspartic acid (L240D),
asparagine (L240N), or tyrosine (L240Y); (dd) the arginine residue at a
position corresponding
to position 241 of wild-type human Neu2 is substituted by alanine (R24 I A),
aspartic acid
(R241D), leucine (R241L), glutamine (R241Q), or tyrosine (R241Y); (ee) the
alanine residue at
a position corresponding to position 242 of wild-type human Neu2 is
substituted by cysteine
(A242C), phenylalanine (A242F), glycine (A242G), histidine (A242H), isoleucine
(A242I),
lysine (A242K), leucine (A242L), methionine (A242M), asparagine (A242N),
glutamine
(A242Q), arginine (A242R), serine (A242S), valine (A242V), tryptophan (A242W),
or tyrosine
(A242Y); (if) the valine residue at a position corresponding to position 244
of wild-type human
Neu2 is substituted by isoleucine (V244I), lysine (V244K), or proline (V244P);
(gg) the
glutamic acid residue at a position corresponding to position 257 of wild-type
human Neu2 is
substituted by proline (E257P); (hh) the serine residue at a position
corresponding to position
258 is substituted by cysteine (S258C); (ii) the leucine residue at a position
corresponding to
position 260 of wild-type human Neu2 is substituted by aspartic acid (L260D),
phenylalanine
(L260F), glutamine (L260Q), or threonine (L260T); (jj) the valine residue at a
position
corresponding to position 265 of wild-type human Neu2 is substituted by
phenylalanine
(V265F); (kk) the glutamine residue at a position corresponding to position
270 of wild-type
human Neu2 is substituted by alanine (Q270A), histidine (Q270H), phenylalanine
(Q270F),
proline (Q270P), serine (Q270S), or threonine (Q270T); (11) the tryptophan
residue at a position
corresponding to position 292 of wild-type human Neu2 is substituted by
arginine (W292R);
(mm) the serine residue at a position corresponding to position 301 of wild-
type human Neu2 is
substituted by alanine (S301A), aspartic acid (S301D), glutamic acid (S301E),
phenylalanine
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(S301F), glycine (S301G), histidine (S301H), isoleucine (S3011), lysine
(S301K), leucine
(S301L), methionine (S301M), asparagine (S301N), proline (S301P), glutamine
(S301Q),
arginine (S301R), threonine (S301T), valine (S301V), tryptophan (S301W), or
tyrosine
(S301Y), (nn) the tryptophan residue at a position corresponding to position
302 of wild-type
human Neu2 is substituted by alanine (W302A), aspartic acid (W302D), glutamic
acid (W302E),
phenylalanine (W302F), glycine (W302G), histidine (W302H), isoleucine (W3021),
lysine
(W302K), leucine (W302L), methionine (W302M), asparagine (W302N), proline
(W302P),
glutamine (W302Q), arginine (W302R), serine (W302S), threonine (W302T), valine
(W302V),
or tyrosine (W302Y); (oo) the valine residue at a position corresponding to
position 363 of wild-
type human Neu2 is substituted by arginine (V363R); or (pp) the leucine
residue at a position
corresponding to position 365 of wild-type human Neu2 is substituted by
glutamine (L365Q),
histidine (L365H), isoleucine (L365I), lysine (L365K) or serine (L365S); or
the sialidase
comprises a combination of any of the foregoing substitutions For example, the
sialidase may
comprise a substitution selected from K9D, A42R, P62G, P62N, P62S, P62T, D8OP,
A93E,
QI26H, Q I 26Y, R 1 89P, H239P, A242T, Q270A, Q270S, Q270T, S30 I A, S30IR,
W302K,
W302R, V363R, and L365I, or a combination of any of the foregoing
substitutions.
102541 In certain embodiments, the recombinant mutant human sialidase
comprises a deletion of
leucine residue at a position corresponding to position 184 of wild-type human
Neu2 (AL184),
a deletion of a histidine residue at a position corresponding to position 185
of wild-type human
Neu2 (AH185), a deletion of a proline residue at a position corresponding to
position 186 of
wild-type human Neu2 (AP186), a deletion of an isoleucine residue at a
position corresponding
to position 187 of wild-type human Neu2 (AI187), and a deletion of a glutamine
residue at a
position corresponding to position 184 of wild-type human Neu2 (AQ188), or a
combination of
any of the foregoing deletions.
102551 In certain embodiments, the recombinant mutant human sialidase
comprises an insertion
between a threonine residue at a position corresponding to position 216 of
wild-type human
Neu2 and a leucine residue at a position corresponding to position 217 of wild-
type human
Neu2, for example, an insertion of an amino acid selected from S, T, Y, L, F,
A, P, V, I, N, D,
and H.
102561 Additional exemplary sialidase mutations, and combinations of sialidase
mutations, are
described in International (PCT) Patent Application Publication No. WO
2019/136167,
including in the Detailed Description in the section entitled "I. Recombinant
Human Sialidases,"
and in the Examples in Examples 1, 2, 3, 4, 5, and 6, and International (PCT)
Patent Application
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Publication No. WO 2021/003469, including in the Detailed Description in the
section entitled
"I. Recombinant Human Sialidases," and in the Examples in Examples 2, 3, 4,
and 5, and in
International (PCT) Patent Application No. PCT/US2021/040240, filed July 2,
2021, including
in the Detailed Description in the section entitled "I. Recombinant Human
Sialidases," and in the
Examples in Examples 2, 3, 4, and 5.
6. Combinations of Substitutions
102571 In certain embodiments, the recombinant mutant human sialidase
comprises a
combination of any of the mutations contemplated herein. For example, the
recombinant mutant
sialidase enzyme may comprise a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15 or
more of the mutations contemplated herein. It is contemplated that the
recombinant mutant
sialidase enzyme may comprise 1-15, 1-10, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15,
2-10, 2-7, 2-6, 2-5,
2-4, 2-3, 3-15, 3-10, 3-7, 3-6, 3-5, or 3-4 of the mutations contemplated
herein.
102581 For example, the recombinant mutant sialidase enzyme may comprise a M1
deletion
(AM1), MIA substitution, M1D substitution, V6Y substitution, K9D substitution,
P62G
substitution, P62N substitution, P62S substitution, P62T substitution, A93E
substitution, 1187K
substitution, Q270A substitution, S301R substitution, W3 02K substitution,
C332A substitution,
V363R substitution, L365I substitution, or a combination of any of the
foregoing.
102591 In certain embodiments, the recombinant mutant sialidase enzyme
comprises a M1
deletion (AM1), MIA substitution, M1D substitution, V6Y substitution, I187K
substitution,
C332A substitution, or a combination of any of the foregoing. For example, the
recombinant
mutant sialidase enzyme may comprise a combination of mutations selected from:
MIA and
V6Y; M1A and I187K; MIA and C332A; M1D and V6Y; M1D and I187K; M1D and C332A;
AM1 and V6Y; AM1 and I187K; AM1 and C332A; V6Y and I187K; V6Y and C332A; I187K
and C332A; M1A, V6Y, and I187K; M1A, V6Y, and C332A; MIA, I187K, and C332A;
M1D,
V6Y, and I187K; MID, V6Y, and C332A; MID, I187K, and C332A; AM1, V6Y, and
I187K;
AM1, V6Y, and C332A; AM1, I187K, and C332A; V6Y, I187K, and C332A; MIA, V6Y,
I187K, and C332A; M1D, V6Y, I187K, and C332A; and AM1, V6Y, I187K, and C332A.
102601 In certain embodiments, the recombinant mutant sialidase enzyme
comprises (i) an
amino acid substitution identified in TABLE 7, or a combination of any amino
acid substitutions
identified in TABLE 7, and (ii) an MI deletion (AM1), MIA substitution, MID
substitution,
V6Y substitution, 1187K substitution, C332A substitution, or a combination of
any of the
foregoing. For example, the recombinant mutant sialidase enzyme may comprise
(i) an amino
acid substitution identified in TABLE 7, or a combination of any amino acid
substitutions
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identified in TABLE 7, and (ii) a combination of mutations selected from: MIA
and V6Y; MIA
and I187K; WA and C332A; M1D and V6Y; M1D and I187K; M1D and C332A; AM1 and
V6Y; AM1 and I187K; AM1 and C332A; V6Y and I187K; V6Y and C332A; I187K and
C332A;
M1A, V6Y, and I187K; M1A, V6Y, and C332A; M1A, I187K, and C332A; M1D, V6Y, and
I187K; M1D, V6Y, and C332A; M1D, I187K, and C332A; AM1, V6Y, and I187K; AM1,
V6Y,
and C332A; AM1, I187K, and C332A; V6Y, I187K, and C332A; M1A, V6Y, I187K, and
C332A; M1D, V6Y, I187K, and C332A; and AM1, V6Y, I187K, and C332A.
102611 In certain embodiments, the recombinant mutant sialidase enzyme
comprises: (a) the
M1D, V6Y, P62G, A93E, I187K, and C332A substitutions; (b) the M1D, V6Y, K9D,
A93E,
I187K, C332A, V363R, and L365I substitutions; (c) the M1D, V6Y, P62N, I187K,
and C332A
substitutions; (d) the M1D, V6Y, I187K, Q270A, S301R, W302K, and C332A
substitutions; (e)
the M1D, V6Y, P62S, I187K, Q270A, S301R, W302K, and C332A substitutions; (f)
the M1D,
V6Y, P62T, 1187K, Q270A, S301R, W302K, and C332A substitutions; (g) the M1D,
V6Y,
P62N, I187K, Q270A, S301R, W302K, and C332A substitutions; (h) the M1D, V6Y,
P62G,
A93E, I187K, S301A, W302R, and C332A substitutions; (i) the M1D, V6Y, P62G,
A93E,
Q126Y, I187K, Q270T, and C332A substitutions; (j) the M1D, V6Y, P62G, A93E,
Q126Y,
I187K, and C332A substitutions; (k) the M1D, V6Y, P62G, A93E, Q126Y, I187K,
A242F,
Q270T, and C332A substitutions; or (1) the M1D, V6Y, A42R, P62G, A93E, Q126Y,
I187K,
A242F, Q270T, and C332A mutations.
102621 In certain embodiments, the recombinant mutant human sialidase
comprises a
substitution of a senile residue at a position corresponding to position 301
of wild-type human
Neu2 (S301) in combination with a substitution of a tryptophan residue at a
position
corresponding to position 302 of wild-type human Neu2 (W302). For example, the
recombinant
mutant human sialidase may comprise a combination of substitutions
corresponding to a
combination of substitutions listed in a row of TABLE 8 (amino acid positions
corresponding to
wild-type human Neu2 (SEQ ID NO: 1)). For example, the recombinant mutant
human sialidase
may comprise: the S301K and W302R substitutions; the S301K and W302K
substitutions; or the
S301A and W302S substitutions.
TABLE 8
Substitutions
S301A, W302R
S301A, W302S
S301A, W302T
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Substitutions
S301K, W302S
S301N, W302S
S301T, W302S
S301T, W302T
S301T, W302R
S301A, W302A
S301K, W302R
S30IK, W302T
S301N, W302T
S301K, W302K
S30IP, W302R
S301P, W302S
S301P, W302T
102631 In certain embodiments, the recombinant mutant human sialidase
comprises a
combination of substitutions corresponding to a combination of substitutions
listed in a row of
TABLE 9 (amino acid positions corresponding to wild-type human Neu2 (SEQ IT)
NO. 1))
TABLE 9
Substitutions
M1D, V6Y, P62G, I187K, C332A
M1D, V6Y, K9D, I187K, C332A, V363R, L365I
M1D, V6Y, P62G, A93E, I187K, C332A
MID, V6Y, K9D, I187K, C332A, V363R, L365K
MID, V6Y, K9D, I187K, C332A, V363R, L365S
MID, V6Y, K9D, I187K, C332A, V363R, L365Q
M1D, V6Y, K9D, I187K, C332A, V363R, L365H
MID, V6Y, A93K, I187K, C332A
MID, V6Y, A93E, I187K, C332A
V6Y, I187K, W292R
V6Y, G107D, I187K
V6Y, C125L
C125L, I187K
V6Y, C125L, I187K
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Substitutions
M1D, V6Y, K45A, I187K, C332A
M1D, V6Y, Q270A, I187K, C332A
MID, V6Y, K44R, K45R, I187K, C332A
MID, V6Y, Q112R, I187K, C332A
M1D, V6Y, Q270F, Il 87K, C332A
MID, V6Y, I187K, S301R, W302K, C332A
MID, V6Y, K44E, K45E, I187K, C332A
MID, V6Y, II87K, L2I7V, C332A
M1D, V6Y, I187K, L217A, C332A
M1D, V6Y, K44E, K45E, I187K, S301R, W302K, C332A
MID, V6Y, Q112R, II87K, S301R, W302K, C332A
M1D, V6Y, I187K, Q270A, S301R, W302K, C332A
MID, V6Y, K44E, K45E, Q112R, I187K, C332A
MID, V6Y, K44E, K45E, I187K, Q270A, C332A
MID, V6Y, K45A, I187K, Q270A, C332A
M1D, V6Y, I187K, Q270H, C332A
MID, V6Y, I187K, Q270P, C332A
M1D, V6Y, Q112K, I187K, C332A
M1D, V6Y, P62S, I187K, Q270A, S301R, W302K, C332A
M1D, V6Y, P62T, I187K, Q270A, S301R, W302K, C332A
MID, V6Y, P62N, I187K, Q270A, S301R, W302K, C332A
V6Y, P62H, I187K
V6Y, Q108H, I187K
MID, V6Y, P62H, Ii 87K, C332A
MID, V6Y, P62G, I187K, C332A
V6Y, P62G, I187K
M1D, V6Y, P62H, I187K
MID, V6Y, Q108H, I187K
MID, V6Y, P62N, I187K, C332A
MID, V6Y, P62D, I187K, C332A
M1D, V6Y, P62E, I187K, C332A
V6Y, C164G, I187K, T249A
V6Y, C164G, I187K
V6Y, Q126L, I187K D251G
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Substitutions
V6Y, L54M, Q69H, R78K, A171G, I187K
V6Y, P62T, I187K
V6Y, A150V, I187K
P5H, V6Y, P62S, I187K
V6Y, C164G, I187K
Q126Y, Q170T
Q126Y, A242F, Q2701
MID, V6Y, P62G, A93E, Q126E, I187K, C332A
M1D, V6Y, P62G, A93E, Q126I, I187K, C332A
M1D, V6Y, P62G, A93E, Q126L, I187K, C332A
MID, V6Y, P62G, A93E, Q126Y, I187K, C332A
M1D, V6Y, P62G, A93E, Q126F, I187K, C332A
MID, V6Y, P62G, A93E, Q126H, I187K, C332A
M1D, V6Y, P62G, A93E, I187K, Q270S, C332A
M1D, V6Y, P62G, A93E, I187K, Q270T, C332A
MID, V6Y, P62G, A93E, Q126Y, I187K, Q270T, C332A
M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, C332A
MID, V6Y, P62G, D8OP, A93E, I187K, C332A
M1D, V6Y, P62G, A93E, R170P, I187K, C332A
MID, V6Y, P62G, A93E, Ti 87K, Q1 88P, C332A
MID, V6Y, P62G, A93E, 1187K, R189P, C332A
M1D, V6Y, P62G, A93E, I187K, E225P, C332A
M1D, V6Y, P62G, A93E, 1187K, H239P, C332A
MID, V6Y, P62G, A93E, I187K, E257P, C332A
M1D, V6Y, P62G, A93E, I187K, S301A, C332A
M1D, V6Y, P62G, A93E, I187K, S301D, C332A
M1D, V6Y, P62G, A93E, I187K, S301E, C332A
MID, V6Y, P62G, A93E, I187K, S301F, C332A
M1D, V6Y, P62G, A93E, I187K, S301H, C332A
M1D, V6Y, P62G, A93E, I187K, S301K, C332A
M1D, V6Y, P62G, A93E, I187K, S301L, C332A
MID, V6Y, P62G, A93E, I187K, S301M, C332A
M1D, V6Y, P62G, A93E, I187K, S301N, C332A
MID, V6Y, P62G, A93E, I187K, S301P, C332A
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Substitutions
MID, V6Y, P62G, A93E, I187K, S301Q, C332A
MID, V6Y, P62G, A93E, I187K, S301R, C332A
MID, V6Y, P62G, A93E, I187K, S301T, C332A
MID, V6Y, P62G, A93E, I187K, S301V, C332A
MID, V6Y, P62G, A93E, I187K, S301W, C332A
MID, V6Y, P62G, A93E, I187K, S301Y, C332A
M1D, V6Y, P62G, A93E, I187K, W302A, C332A
MID, V6Y, P62G, A93E, I187K, W302D, C332A
MID, V6Y, P62G, A93E, I187K, W302F, C332A
M1D, V6Y, P62G, A93E, I187K, W302G, C332A
MID, V6Y, P62G, A93E, I187K, W302H, C332A
MID, V6Y, P62G, A93E, I187K, W3021, C332A
MILD, V6Y, P62G, A93E, I187K, W302L, C332A
MID, V6Y, P62G, A93E, I187K, W302M, C332A
M1D, V6Y, P62G, A93E, I187K, W302N, C332A
MID, V6Y, P62G, A93E, I187K, W302P, C332A
M1D, V6Y, P62G, A93E, I187K, W302Q, C332A
MILD, V6Y, P62G, A93E, I187K, W302R, C332A
MID, V6Y, P62G, A93E, I187K, W302S, C332A
M1D, V6Y, P62G, A93E, Ti 87K, W302T, C332A
MI D, V6Y, P62G, A93E, I187K, W302V, C332A
M1D, V6Y, P62G, A93E, I187K, W302Y, C332A
M113, V6Y, P62G, A93E, 1187K, S301A, W302A, C332A
MID, V6Y, P62G, A93E, I187K, S30 IA, W302R, C332A
MID, V6Y, P62G, A93E, I187K, S301A, W302S, C332A
MID, V6Y, P62G, A93E, I187K, S301A, W302T, C332A
MID, V6Y, P62G, A93E, I187K, S301K, W302S, C332A
MID, V6Y, P62G, A93E, I187K, S301K, W302R, C332A
MID, V6Y, P62G, A93E, I187K, S301K, W302T, C332A
MID, V6Y, P62G, A93E, I187K, S301N, W302S, C332A
MID, V6Y, P62G, A93E, I187K, S301N, W302T, C332A
MID, V6Y, P62G, A93E, I187K, S301T, W302R, C332A
Q126Y, Q270T
Q126Y, A242F, Q270T
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Substitutions
M1D, V6Y, A42R, P62G, A93E, Q126Y, I187K, A242F, Q270T, and C332A
102641 In certain embodiments, the recombinant mutant human sialidase
comprises the amino
acid sequence of any one of SEQ ID NOs: 48-62, 94, 97, 100, 126, or 234, or an
amino acid
sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence
identity to any one
of SEQ ID NOs: 48-62, 94, 97, 100, 126, or 234.
102651 In certain embodiments, the recombinant mutant human sialidase
comprises the
amino acid sequence of
X iX2 SX3X4X5LQX6ESVFQS GAHAYRI PAL LYL PGQQSLLAFAEQRX7SX8X9DEHAEL IVX10RRG
DYDAX THQVQWX12AQEVVAQAX13LX14GHRSMNPCPLYDX15QT G T L FL FFIAI PX16X17VTEX
i8QQLQTRANVIRLX19X20VT S TDHGRTWS SPRDLTDAAI GPX21YREWST FAVGPGHX22LQLHDX
23X24RSLVVPAYAYRKLHPX25X26X27P I P SAFX2 s FL SHDHGRTWARGH FVX29 QDTX3
oECQVAEV
X3iTGEQRVVILNARSX32X33X34X35RX36QAQSX37NX30GLDFQX39X40QX4iVKKLX42E PPPX43G
X44QGSVI S FP S PRS GPGSPAQX45LLYTHPTHX46X47QRADLGAYLNPRPPAPEAWSEPX4oLLAK
GSX49AYS DLQSMG T GPDGS PL FGX50LYEANDYEE I X5iFX52MFT LKQAFPAE YL PQ (SEQ ID
NO: 238),
wherein Xi is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr,
Val, or not
present, X2 is Ala or Lys, X3 is Asn or Leu, X4 is Pro or His, X5 is Phe, Trp,
Tyr or Val, X6 is Lys
or Asp, X7 is Ala or Arg, X8 is Lys, Arg, or Glu, X9 is Lys, Ala, Arg, or Glu,
Xio is Leu or Met,
XII is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, X12 is Gln or His, X13 is Arg
or Lys, Xi4 is Asp or
Pro, Xi5 is Ala, Glu or Lys, X16 is Gly or Asp, X17 is Gln or His, Xis is Gln,
Arg, or Lys, X19 is
Ala, Cys, Ile, Ser, Val, or Leu, X20 is Gln, Leu, Glu, Phe, His, Ile, Leu, or
Tyr, X21 is Ala or Val,
X22 is Cys or Gly, X23 is Arg or Pro, X24 is Ala or Gly, X25 is Arg, Ile, or
Lys, X26 is Gln or Pro,
X27 is Arg or Pro, X28 is Ala, Cys, Leu, or Val, X29 is Ala, Cys, Asn, Ser, or
Thr, X30 is Leu, Ala,
or Val, X31 is Glu or Pro, X37 is His or Pro, X33 is Leu, Asp, Asn, or Tyr,
X34 is Arg, Ala, Asp,
Leu, Gln, or Tyr, X35 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Gln, Arg, Ser, Val, Trp,
or Tyr, X36 is Val, Ile, or Lys, X37 is Thr or Ala, X38 is Asp or Gly, X39 is
Glu, Lys, or Pro, X40 is
Ser or Cys, X41 is Leu, Asp, Phe, Gln, or Thr, X42 is Val or Phe, X43 is Gln,
Ala, His, Phe, Pro,
Ser, or Thr, X44 is Cys or Val, X45 is Trp or Arg, X46 is Ser, Arg, Ala, Asp,
Glu, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, X47 is Trp, Lys,
Ala, Asp, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, X48 is Lys
or Val, X49 is Ala,
Cys, Ser, or Val, X50 is Cys, Leu, or Val, X51 is Val or Arg, and X52 is Leu,
Gln, His, Ile, Lys, or
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Ser, and the sialidase comprises at least one mutation relative to wild-type
human Neu2 (SEQ ID
NO: 1).
102661 In certain embodiments, the recombinant mutant human sialidase
comprises the
amino acid sequence of
X iASLPX2LQX3E SVFQS GAHAYRI PALLYLPGQQS LLAFAEQRX 4 S KKDEHAEL IVLRRGDYDAX
5 THQVQWQAQEVVAQARLDGHRSMNPCPLYDX 6QT GIL FL FFIAI PGQVTEQQQLQTRANVTRLC
X7VTSTDHGRTWSSPRDL TDAAIGPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPX8Q
RP I PSAFC FL SHDHGRTWARGHFVAQDTLECQVAEVET GEQRVVTLNARSHLRX9RVQAQS TNDG
LDFQESQLVKKLVEPPPX10GCQGSVI S FPS PRS GPGS PAQWL LYTHP THX1iX 12QRADL GAYLN
PRPPAPEAWS E PVLLAKGS Xi3AYSDLQSMGT GPDGS PL FGCLYEANDYEE I Xi4 FX15MFTLKQA
FPAEYLPQ (SEQ ID NO: 239),
wherein Xi is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr,
Val, or not
present, X2 is Phe, Trp, Tyr or Val, X3 is Lys or Asp, X4 is Arg or Ala, X5 is
Pro, Asn, Asp, His,
Glu, Gly, Ser or Thr, X6 is Ala, Glu, or Lys, X7 is Gln, Leu, Glu, Phe, His,
Ile, Leu, or Tyr, Xg is
Arg, Ile, or Lys, X9 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln,
Arg, Ser, Val, Trp,
or Tyr, Xio is Gln, Ala, His, Phe, Pro, Ser, or Thr, XII is Ser, Arg, Ala,
Asp, Glu, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, X12 is Trp, Lys,
Ala, Asp, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, X13 is
Ala, Cys, Ser, or Val,
X14 ls Val or Arg, and X15 is Leu, Gln, His, Ile, Lys, or Ser, and the
sialidase comprises at least
one mutation relative to wild-type human Neu2 (SEQ ID NO: 1). In certain
embodiments, X1 is
Ala, Asp, Met, or not present, X2 is Tyr or Val, X3 is Lys or Asp, X4 is Arg
or Ala, X5 is Pro,
Asn, Gly, Ser or Thr, X6 is Ala or Glu, X7 is Gln or Tyr, X8 is Ile or Lys, X9
is Ala or Thr, Xio is
Gln, Ala, or Thr, Xiiis Ser, Arg, or Ala, X12 is Trp, Lys, or Arg, X13 is Ala
or Cys, X14 is Val or
Arg, and X15 is Leu or Ile.
102671 In certain embodiments, the recombinant mutant human sialidase
comprises a
conservative substitution relative to a recombinant mutant human sialidase
sequence disclosed
herein. As used herein, the term "conservative substitution" refers to a
substitution with a
structurally similar amino acid. For example, conservative substitutions may
include those
within the following groups: Ser and Cys; Leu, Ile, and Val; Glu and Asp; Lys
and Arg; Phe,
Tyr, and Trp; and Gln, Asn, Glu, Asp, and His. Conservative substitutions may
also be defined
by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM
substitution
matrix (e.g., BLO SUM 62 matrix), or the PAM substitution:p matrix (e.g., the
PAM 250 matrix).
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b. Antibody Portion
102681 In certain embodiments, the fusion protein comprises an immunoglobulin
Fc domain.
As used herein, unless otherwise indicated, the term "immunoglobulin Fc
domain" refers to a
fragment of an immunoglobulin heavy chain constant region which, either alone
or in
combination with a second immunoglobulin Fc domain, is capable of binding to
an Fc receptor.
An immunoglobulin Fc domain may include, e.g., immunoglobulin CH2 and CH3
domains. An
immunoglobulin Fc domain may include, e.g., immunoglobulin CH2 and CH3 domains
and an
immunoglobulin hinge region. Boundaries between immunoglobulin hinge regions,
CH2, and
CH3 domains are well known in the art, and can be found, e.g., in the PROSITE
database
(available on the world wide web at prosite.expasy.org).
102691 In certain embodiments, the immunoglobulin Fc domain is derived from a
human IgGl,
IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, and IgM Fc domain. A single amino acid
substitution
(S228P according to Kabat numbering; designated IgG4Pro) may be introduced to
abolish the
heterogeneity observed in recombinant IgG4 antibody. See Angal, S. et al.
(1993) MOL.
IMMUNOL . 30:105-108.
102701 In certain embodiments, the immunoglobulin Fc domain is derived from a
human IgG1
isotype or another isotype that elicits antibody-dependent cell-mediated
cytotoxicity (ADCC)
and/or complement mediated cytotoxicity (CDC). In certain embodiments, the
immunoglobulin
Fc domain is derived from a human IgG1 isotype (e.g., SEQ ID NO: 31 or SEQ ID
NO: 5).
102711 In certain embodiments, the immunoglobulin Fc domain is derived from a
human IgG4
isotype or another isotype that elicits little or no antibody-dependent cell-
mediated cytotoxicity
(ADCC) and/or complement mediated cytotoxicity (CDC). In certain embodiments,
the
immunoglobulin Fc domain is derived from a human IgG4 isotype.
102721 In certain embodiments, the immunoglobulin Fc domain comprises either a
"knob"
mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, for
heterodimerization with a second
polypeptide (residue numbers according to EU numbering, Kabat, E.A., el al.
(1991) SEQUENCES
OF PROTEINS OF IMMUNOLOGICAL IN ___ FEREST, FIFTH EDITION, U.S. Department of
Health and
Human Services, NIH Publication No. 91-3242). For example, in certain
embodiments, the
immunoglobulin Fc domain is derived from a human IgG1 Fc domain and comprises
a Y407T
mutation (e.g., the immunoglobulin Fc domain comprises SEQ ID NO: 32 or SEQ ID
NO: 92).
In certain embodiments, the immunoglobulin Fc domain is derived from a human
IgG1 Fc
domain and comprises a T366Y mutation (e.g., the second polypeptide comprises
SEQ ID NO:
33 or SEQ ID NO: 93).
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[0273] In certain embodiments, the immunoglobulin Fc domain is modified to
prevent to
glycosylation of the Fc domain. For example, in certain embodiments, the
immunoglobulin Fc
domain is derived from a human IgG1 Fc domain and comprises a mutation at
position N297, for
example, an N297A or N297G mutation (residue numbers according to EU
numbering, Kabat,
E.A., et at., supra). For example, in certain embodiments, the fusion protein
comprises SEQ ID
NO: 222, SEQ ID NO: 225, or SEQ ID NO: 226.
[0274] In certain embodiments, the fusion protein comprises an immunoglobulin
antigen-
binding domain. The inclusion of such a domain may improve targeting of a
fusion protein to a
sialylated cancer cell, e.g., a PD-Li expressing cancer cell, and/or to the
tumor
microenvironment. As used herein, unless otherwise indicated, the term
"immunoglobulin
antigen-binding domain" refers to a polypeptide that, alone or in combination
with another
immunoglobulin antigen-binding domain, defines an antigen-binding site.
Exemplary
immunoglobulin antigen-binding domains include, for example, immunoglobulin
heavy chain
variable region and an immunoglobulin light chain variable region, where the
variable regions
together define an antigen binding site, e.g., an anti-PD-Li antigen binding
site.
[0275] In certain embodiments, the immunoglobulin antigen-binding domain is
derived from
an anti-PD-Li antibody. Exemplary anti-PD-Li antibodies are described, for
example, in U.S.
Patent Nos. 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and
8,217,149. Exemplary
anti-PD-L1 antibodies include, atezolizumab (Tecentriq , Genentech),
durvalumab
(AstraZeneca),1VIED14736, avelumab, CS1001 (CStone Therapeutics), KL-A167, CK-
301
(Checkpoint Therapeutics), TQB2450, KNO35, SHR-1316, STI-A1014, BGB-A333,
MSB2311,
HLX-20 and BMS-936559 by Bristol-Myers Squibb.
[0276] In certain embodiments, the immunoglobulin antigen-binding domain is
derived from
avelumab. The avelumab heavy chain amino acid sequence is depicted in SEQ ID
NO: 63, and
the avelumab light chain amino acid sequence is depicted in SEQ ID NO: 64. The
amino acid
sequence of an exemplary scFy derived from avelumab is depicted in SEQ ID NO:
125.
[0277] In certain embodiments, the immunoglobulin antigen-binding domain is
derived from
an anti-PD-Li antibody disclosed herein, for example, an antibody comprising:
(i) an
immunoglobulin heavy chain variable region comprising the amino acid sequence
of SEQ ID
NO: 164 (PAL769-VH), and an immunoglobulin light chain variable region
comprising the
amino acid sequence of SEQ ID NO: 167 (PAL769-VL); (ii) an immunoglobulin
heavy chain
variable region comprising the amino acid sequence of SEQ ID NO: 199 (h769
VH), and an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
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200 (h769-IF3-VL); (iii) an immunoglobulin heavy chain variable region
comprising the amino
acid sequence of SEQ ID NO: 199 (h769-VH), and an immunoglobulin light chain
variable
region comprising the amino acid sequence of SEQ ID NO: 201 (h769-tm2-VL);
(iv) an
immunoglobulin heavy chain variable region comprising the amino acid sequence
of SEQ ID
NO: 199 (h769 VII), and an immunoglobulin light chain variable region
comprising the amino
acid sequence of SEQ ID NO: 202 (h769-tm3-VL); (v) an immunoglobulin heavy
chain variable
region comprising the amino acid sequence of SEQ ID NO: 199 (h769-VH), and an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
204 (h769.T-VL); (vi) an immunoglobulin heavy chain variable region comprising
the amino
acid sequence of SEQ ID NO: 132 (PAL752-VH), and an immunoglobulin light chain
variable
region comprising the amino acid sequence of SEQ ID NO: 136 (PAL752-VL); (vii)
an
immunoglobulin heavy chain variable region comprising the amino acid sequence
of SEQ ID
NO: 140 (PAL759-VH), and an immunoglobulin light chain variable region
comprising the
amino acid sequence of SEQ ID NO: 144 (PAL759-VL); (viii) an immunoglobulin
heavy chain
variable region comprising the amino acid sequence of SEQ ID NO: 148 (PAL760-
VH), and an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
152 (PAL760-VL); (ix) an immunoglobulin heavy chain variable region comprising
the amino
acid sequence of SEQ ID NO: 156 (PAL767-VH), and an immunoglobulin light chain
variable
region comprising the amino acid sequence of SEQ ID NO: 160 (PAL767-VL); (x)
an
immunoglobulin heavy chain variable region comprising the amino acid sequence
of SEQ ID
NO: 170 (PAL771-VH), and an immunoglobulin light chain variable region
comprising the
amino acid sequence of SEQ ID NO: 174 (PAL771-VL); (xi) an immunoglobulin
heavy chain
variable region comprising the amino acid sequence of SEQ ID NO: 178 (PAL785-
VH), and an
immunoglobulin light chain variable region comprising the amino acid sequence
of SEQ ID NO:
182 (PAL785-VL); (xii) an immunoglobulin heavy chain variable region
comprising the amino
acid sequence of SEQ ID NO: 186 (PAL787-VH), and an immunoglobulin light chain
variable
region comprising the amino acid sequence of SEQ ID NO: 190 (PAL787-VL); or
(xiii) an
immunoglobulin heavy chain variable region comprising the amino acid sequence
of SEQ ID
NO: 194 (PAL788-VH), and an immunoglobulin light chain variable region
comprising the
amino acid sequence of SEQ ID NO: 198 (PAL788-VL)
c. Linker
102781 In certain embodiments, the sialidase portion of the fusion protein can
be linked or
fused directly to the anti-PD-Li antibody portion (e.g., immunoglobulin Fc
domain and/or
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immunoglobulin antigen-binding domain) of the fusion protein. In other
embodiments, the
sialidase portion can be covalently bound to the anti-PD-Li antibody portion
by a linker.
102791 The linker may couple, with one or more natural amino acids, the
sialidase, or
functional fragment thereof, and the antibody portions or fragments, where the
amino acid (for
example, a cysteine amino acid) may be introduced by site-directed
mutagenesis. The linker
may include one or more unnatural amino acids. It is contemplated that, in
certain
circumstances, a linker containing for example, one or more sulfhydryl
reactive groups (e.g., a
maleimide) may covalently link a cysteine in the sialidase portion or the
antibody portion that is
a naturally occurring cysteine residue or is the product of site-specific
mutagenesis.
102801 The linker may be a cleavable linker or a non-cleavable linker.
Optionally or in
addition, the linker may be a flexible linker or an inflexible linker.
102811 The linker should be a length sufficiently long to allow the sialidase
and the antibody
portions to be linked without steric hindrance from one another and
sufficiently short to retain
the intended activity of the fusion protein. The linker preferably is
sufficiently hydrophilic to
avoid or minimize instability of the fusion protein The linker preferably is
sufficiently
hydrophilic to avoid or minimize insolubility of the fusion protein. The
linker should be
sufficiently stable in vivo (e.g., it is not cleaved by serum, enzymes, etc.)
to permit the fusion
protein to be operative in vivo.
102821 The linker may be from about 1 angstroms (A) to about 150 A in length,
or from about
1 A to about 120 A in length, or from about 5 A to about 110 A_ in length, or
from about 10 A to
about 100 A in length. The linker may be greater than about 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 25, 27, 30 or greater angstroms in length and/or
less than about 110,
100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 43, 42, 41, 40, 39, 38, 37, 36,
35, 34, 33, 32, 31, or
fewer A in length. Furthermore, the linker may be about 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 110, and 120 A in length.
102831 In certain embodiments, the linker comprises a polypeptide linker that
connects or
fuses the sialidase portion of the fusion protein to the anti-PD-Li antibody
portion (e.g.,
immunoglobulin Fc domain and/or immunoglobulin antigen-binding domain) of the
fusion
protein. For example, it is contemplated that a gene encoding a sialidase
portion linked directly
or indirectly (for example, via an amino acid containing linker) to an
antibody portion can be
created and expressed using conventional recombinant DNA technologies. For
example, the
amino terminus of a sialidase portion can be linked to the carboxy terminus of
either the light or
the heavy chain of an antibody portion. For example, for a Fab fragment, the
amino terminus or
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carboxy terminus of the sialidase can be linked to the first constant domain
of the heavy
antibody chain (CH1). When a linker is employed, the linker may comprise
hydrophilic amino
acid residues, such as Gln, Ser, Gly, Glu, Pro, His and Arg. In certain
embodiments, the linker is
a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15
amino acid
residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid
residues, 4-20
amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25
amino acid
residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino
acid residues.
Exemplary linkers include glycine and serine-rich linkers, e.g., (GlyGlyPro),
or
(GlyGlyGlyGlySer)., where n is 1-5. In certain embodiments, the linker
comprises, consists, or
consists essentially of GGGGS (SEQ ID NO: 121). In certain embodiments, the
linker
comprises, consists, or consists essentially of GGGGSGGGGS (SEQ ID NO: 90). In
certain
embodiments, the linker comprises, consists, or consists essentially of EPKSS
(SEQ ID NO: 91).
Additional exemplary linker sequences are disclosed, e.g., in George et al.
(2003) PROTEIN
ENGINEERING 15:871-879, and U.S. Patent Nos. 5,482,858 and 5,525,491.
102841 In certain embodiments, the fusion protein comprises the amino acid
sequence of any
one of SEQ ID NOs: 65-75, 78, 81-89, 95, 96, 98, 99, 101, 102, 104, 106, 108,
110, 112, 114,
122-124, 127, 128, 205-207, 211, 213, 214, or 219, or an amino acid sequence
that has at least
85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID
NOs. 65-75,
78, 81-89, 95, 96, 98, 99, 101, 102, 104, 106, 108, 110, 112, 114, 122-124,
127, 128, 205-207,
211, 213, 214, or 219.
d. Antibody Conjugates
102851 The invention further provides antibody conjugates containing one or
more of the
fusion proteins disclosed herein. As used herein, unless otherwise indicated,
the term "antibody
conjugate- is understood to refer to an antibody, or a functional fragment
thereof, that comprises
antigen-binding activity (e.g., anti-PD-Li antigen-binding activity) and/or Fe
receptor-binding
activity, conjugated (e.g., covalently coupled) to an additional functional
moiety. In certain
embodiments, the antibody or functional antibody fragment is conjugated to a
sialidase enzyme,
e.g., a recombinant mutant human sialidase enzyme disclosed herein. In certain
embodiments,
an antibody conjugate comprises a single polypeptide chain. In certain
embodiments, an
antibody conjugate comprises two, three, four, or more polypeptide chains that
are covalently or
non-covalently associated together to produce a multimeric complex, e.g., a
dimeric, trimeric or
tetrameric complex. For example, an antibody conjugate may comprise a first
polypeptide
(fusion protein) comprising a recombinant mutant human sialidase enzyme and an
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immunoglobulin heavy chain, and a second polypeptide comprising an
immunoglobulin light
chain, where, for example, the immunoglobulin heavy and light chains together
define a single
antigen-binding site, e.g., an anti-PD-Li antigen-binding site.
102861 In certain embodiments, the antibody conjugate can include a single
sialidase. In other
embodiments, the antibody conjugate can include more than one (e.g., two)
sialidases. If more
than one sialidase is included, the sialidases can be the same or different In
certain
embodiments, the antibody conjugate can include a single anti-PD-Li antigen-
binding site. In
other embodiments, the antibody conjugate can include more than one (e.g.,
two) anti-PD-Li
antigen-binding sites. If two antigen-binding sites are used, they can be the
same or different. In
certain embodiments, the antibody conjugate comprises an immunoglobulin Fc
fragment.
102871 In certain embodiments, the antibody conjugate comprises one or two
immunoglobulin
heavy chains, or a functional fragment thereof In certain embodiments, the
antibody conjugate
comprises one or two immunoglobulin light chains, or a functional fragment
thereof. In certain
embodiments, the antibody conjugate comprises a sialidase fused to the N- or C-
terminus of an
immunoglobulin heavy chain or an immunoglobulin light chain.
102881 FIGURE 4 depicts exemplary antibody conjugate constructs containing one
or more
sialidase enzymes. For example, in FIGURE 4A, a first anti-PD-Li antigen-
binding site (e.g.,
defined by a Vu and VL domains) is depicted as 10, a second anti-PD-Li antigen-
binding site is
depicted as 20, a sialidase is depicted as 30, and a Fe is depicted as 40. In
each of the constructs
depicted in FIGUREs 4A-4I it is understood that the Fe may optionally be
modified in some
manner, e.g., using Knobs-into-Holes type technology, e.g., as depicted by 50
in FIGURE 4B.
Throughout FIGURE 4 similar structures are depicted by similar schematic
representations.
102891 FIGURE 4A depicts antibody conjugate constructs comprising a
first polypeptide
comprising a first immunoglobulin light chain; a second polypeptide comprising
a first
immunoglobulin heavy chain; a third polypeptide comprising a second
immunoglobulin heavy
chain; and a fourth polypeptide comprising a second immunoglobulin light
chain. The first and
second polypeptides can be covalently linked together, the third and fourth
polypeptides can be
covalently linked together, and the second and third polypeptides can be
covalently linked
together. The covalent linkages can be disulfide bonds. In certain
embodiments, the first
polypeptide and the second polypeptide together define a first anti-PD-Li
antigen-binding site as
depicted as 10, and the third polypeptide and the fourth polypeptide together
define a second
anti-PD-Li antigen-binding site as depicted as 20. A sialidase enzyme as
depicted as 30 can be
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conjugated to the N- or C-terminus of the first and second immunoglobulin
light chain or the
first and second immunoglobulin heavy chain.
102901 FIGURE 4B depicts antibody conjugate constructs comprising a
first polypeptide
comprising a first immunoglobulin light chain; a second polypeptide comprising
a first
immunoglobulin heavy chain; a third polypeptide comprising a second
immunoglobulin heavy
chain; and a fourth polypeptide comprising a second immunoglobulin light chain
The first and
second polypeptides can be covalently linked together, the third and fourth
polypeptides can be
covalently linked together, and the second and third polypeptides can be
covalently linked
together. The covalent linkages can be disulfide bonds In certain embodiments,
the first
polypeptide and the second polypeptide together define a first anti-PD-Li
antigen-binding site,
and the third polypeptide and the fourth polypeptide together define a second
anti-PD-Li
antigen-binding site. A sialidase enzyme can be conjugated to the N- or C-
terminus of the first
immunoglobulin light chain or the first immunoglobulin heavy chain
102911 FIGURE 4C depicts antibody conjugate constructs comprising a
first polypeptide
comprising an immunoglobulin light chain; a second polypeptide comprising an
immunoglobulin heavy chain; and a third polypeptide comprising an
immunoglobulin Fc
domain. The first and second polypeptides can be covalently linked together
and the second and
third polypeptides can be covalently linked together. The covalent linkages
can be disulfide
bonds. In certain embodiments, the first polypeptide and the second
polypeptide together define
an anti-PD-L1 antigen-binding site. A sialidase enzyme can be conjugated to
the N- or C-
terminus of the first immunoglobulin light chain or the first immunoglobulin
heavy chain.
102921 FIGURE 4D depicts antibody conjugate constructs comprising a
first polypeptide
comprising an immunoglobulin light chain; a second polypeptide comprising an
immunoglobulin heavy chain; and a third polypeptide comprising an
immunoglobulin Fc domain
and a first sialidase enzyme. The first and second polypeptides can be
covalently linked together
and the second and third polypeptides can be covalently linked together. The
covalent linkages
can be disulfide bonds. The third polypeptide comprises the sialidase and the
immunoglobulin
Fc domain in an N- to C-terminal orientation. In certain embodiments, the
first polypeptide and
the second polypeptide together define an anti-PD-Li antigen-binding site. An
optional second
sialidase enzyme can be conjugated to the N- or C-terminus of the first
immunoglobulin light
chain or the first immunoglobulin heavy chain.
102931 FIGURE 4E depicts antibody conjugate constructs comprising a
first polypeptide
comprising an immunoglobulin light chain; a second polypeptide comprising an
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immunoglobulin heavy chain; and a third polypeptide comprising an
immunoglobulin Fc domain
and a first sialidase enzyme. The first and second polypeptides can be
covalently linked together
and the second and third polypeptides can be covalently linked together. The
covalent linkages
can be disulfide bonds. The third polypeptide comprises the immunoglobulin Fc
domain and the
sialidase in an N- to C-terminal orientation. In certain embodiments, the
first polypeptide and
the second polypeptide together define an anti-PD-Li antigen-binding site. An
optional second
sialidase enzyme can be conjugated to the N- or C-terminus of the first
immunoglobulin light
chain or the first immunoglobulin heavy chain.
102941 FIGURE 4F depicts antibody conjugate constructs comprising a
first polypeptide
comprising a first immunoglobulin Fc domain, and a second polypeptide
comprising a second
immunoglobulin Fc domain. The first and second polypeptides can be covalently
linked
together. The covalent linkages can be disulfide bonds. A sialidase enzyme can
be conjugated
to the N- or C-terminus of the first immunoglobulin Fc domain or to the N- or
C-terminus of the
second immunoglobulin Fc domain. An optional second sialidase enzyme can be
conjugated to
the N- or C-terminus of the first immunoglobulin Fc domain or to the N- or C-
terminus of the
second immunoglobulin Fc domain.
102951 FIGURE 4G depicts antibody conjugate constructs comprising a
first polypeptide
comprising an immunoglobulin light chain; and a second polypeptide comprising
an
immunoglobulin heavy chain variable region. The first and second polypeptides
can be
covalently linked together. The covalent linkages can be disulfide bonds. In
certain
embodiments, the first polypeptide and the second polypeptide together define
an anti-PD-Li
antigen-binding site. The sialidase enzyme can be conjugated to the N- or C-
terminus of the
immunoglobulin light chain or the immunoglobulin heavy chain variable region.
102961 FIGURE 411 depicts antibody conjugate constructs comprising a
first polypeptide
comprising a first immunoglobulin Fc domain, and a second polypeptide
comprising a second
immunoglobulin Fc domain. The first and second polypeptides can be covalently
linked
together. The covalent linkages can be disulfide bonds. A sialidase enzyme can
be conjugated
to the N-terminus of the first immunoglobulin Fc domain or the second
immunoglobulin Fc
domain. An optional second sialidase enzyme can be conjugated to the N-
terminus of the
second immunoglobulin Fc domain or the first immunoglobulin Fc domain,
respectively. A
single chain variable fragment (scFv) can be conjugated to the C-terminus of
the first
immunoglobulin Fc domain or the second immunoglobulin Fc domain. An optional
second
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single chain variable fragment (scFv) can be conjugated to the C-terminus of
the first
immunoglobulin Fc domain or the second immunoglobulin Fc domain, respectively.
102971 FIGURE 41 depicts antibody conjugate constructs similar to
those depicted in
FIGURE 411 except that each scFv is replaced with an immunoglobulin antigen
binding
fragment, e.g-., a Fab For example, FIGURE 41 depicts antibody conjugate
constructs
comprising a first polypeptide comprising a first immunoglobulin Fc domain,
and a second
polypeptide comprising a second immunoglobulin Fc domain. The first and second
polypeptides
can be covalently linked together. The covalent linkages can be disulfide
bonds. A sialidase
enzyme can be conjugated to the N-terminus of the first immunoglobulin Fc
domain or the
second immunoglobulin Fc domain. An optional second sialidase enzyme can be
conjugated to
the N-terminus of the second immunoglobulin Fc domain or the first
immunoglobulin Fc
domain, respectively. An antibody fragment (Fab) can be conjugated or fused to
the C-terminus
of the first immunoglobulin Fc domain or the second immunoglobulin Fc domain.
An optional
second antibody fragment (Fab) can be conjugated or fused to the C-terminus of
the second
immunoglobulin Fc domain or the first immunoglobulin Fc domain, respectively.
In the case of
a fusion, the C terminus of the Fc domain is linked (either by a bond or an
amino acid linker) to
a first polypeptide chain defining an anti-PD-Li immunoglobulin antigen
binding fragment. In
the case of antibodies that have an antigen binding site defined by a single
variable region, then
this may be sufficient to impart binding affinity to a target antigen, e.g.,
PD-Li. In other
instances, e.g., in the case of a human antibody, the first polypeptide chain
defining an
immunoglobulin antigen binding fragment can be conjugated (e.g., covalently
conjugated, e.g.,
via a disulfide bond) to a second polypeptide chain defining an immunoglobulin
antigen binding
fragment, there the two antigen binding fragments together define an antigen
binding site for
binding the target antigen, e.g., PD-Li.
102981 FIGURE 5 depicts additional antibody conjugate constructs. For example,
FIGURE
5 depicts an antibody conjugate construct comprising a first polypeptide
comprising an
immunoglobulin light chain; a second polypeptide comprising an immunoglobulin
heavy chain
and an scFv; and a third polypeptide comprising an immunoglobulin Fc domain
and a first
sialidase enzyme. The first and second polypeptides can be covalently linked
together and the
second and third polypeptides can be covalently linked together. The covalent
linkages can be
disulfide bonds. The second polypeptide comprises the heavy chain and the scFv
in an N- to C-
terminal orientation. The third polypeptide comprises the sialidase and the
immunoglobulin Fc
domain in an N- to C-terminal orientation. In certain embodiments, the first
polypeptide and the
second polypeptide together define a first antigen-binding site. In certain
embodiments, the
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scFv defines a second antigen-binding site. FIGURE 5 depicts an additional
antibody construct
comprising a first polypeptide comprising an immunoglobulin light chain; a
second polypeptide
comprising an immunoglobulin heavy chain; and a third polypeptide comprising
an
immunoglobulin Fc domain and a first sialidase enzyme, wherein a Fab fragment
is conjugated
to the N-terminus of the immunoglobulin heavy chain. The first and second
polypeptides can be
covalently linked together and the second and third polypeptides can be
covalently linked
together. The covalent linkages can be disulfide bonds. The third polypeptide
comprises the
sialidase and the immunoglobulin Fc domain in an N- to C-terminal orientation.
In certain
embodiments, the first polypeptide and the second polypeptide together define
a first antigen-
binding site. In certain embodiments, the Fab fragment defines a second
antigen-binding site.
In each of the constructs depicted in FIGURE 5 it is understood that an scFv,
when present,
may be replaced with a Fab fragment, or a Fab fragment, when present, may be
replaced with an
scFv. In each of the constructs depicted in FIGURE 5, it is understood that
the Fc may
optionally be modified in some manner.
102991 In certain embodiments, the antibody conjugate comprises a first
polypeptide
comprising a first immunoglobulin light chain; a second polypeptide comprising
a first
immunoglobulin heavy chain and a first sialidase; a third polypeptide
comprising a second
immunoglobulin heavy chain and a second sialidase, and a fourth polypeptide
comprising a
second immunoglobulin light chain. An example of this embodiment is shown in
FIGURE 6A.
The first and second polypeptides can be covalently linked together, the third
and fourth
polypeptides can be covalently linked together, and the second and third
polypeptides can be
covalently linked together. The covalent linkages can be disulfide bonds. In
certain
embodiments, the first polypeptide and the second polypeptide together define
a first anti-PD-Li
antigen-binding site, and the third polypeptide and the fourth polypeptide
together define a
second anti-PD-Li antigen-binding site. In certain embodiments, the second and
third
polypeptides comprise the first and second immunoglobulin heavy chain and the
first and second
sialidase, respectively, in an N- to C-terminal orientation. In certain
embodiments, the second
and third polypeptides comprise the first and second sialidase and the first
and second
immunoglobulin heavy chain, respectively, in an N- to C-terminal orientation.
103001 In certain embodiments, the antibody conjugate comprises a first
polypeptide
comprising an immunoglobulin light chain; a second polypeptide comprising an
immunoglobulin heavy chain; and a third polypeptide comprising an
immunoglobulin Fc domain
and a sialidase. An example of this embodiment is shown in FIGURE 6B. The
first and second
polypeptides can be covalently linked together and the second and third
polypeptides can be
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covalently linked together. The covalent linkages can be disulfide bonds. In
certain
embodiments, the first polypeptide and the second polypeptide together define
an anti-PD-L1
antigen-binding site. In certain embodiments, the third polypeptide comprises
the sialidase and
the immunoglobulin Fc domain in an N- to C-terminal orientation, or the
immunoglobulin Fe
domain and the sialidase in an N- to C-terminal orientation.
103011 In certain embodiments, the first polypeptide comprises the amino acid
sequence of any
one of SEQ ID NOs: 65 or 205, or an amino acid sequence that has at least 85%,
90%, 95%,
96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 65 or 205.
In certain
embodiments, the second polypeptide comprises the amino acid sequence of any
one of SEQ ID
NOs: 66, 104, 124, 206, or 213, or an amino acid sequence that has at least
85%, 90%, 95%,
96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 66, 104,
124, 206, or
213. In certain embodiments, the third polypeptide comprises the amino acid
sequence of any
one of SEQ ID NOs: 67-73, 78, 81-87, 95, 96, 98, 99, 101, 102, 106, 108, 112,
122, 123, 127,
128, 207, 211, 214, or 219, or an amino acid sequence that has at least 85%,
90%, 95%, 96%,
97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 67-73, 78, 81-87,
95, 96, 98,
99, 101, 102, 106, 108, 112, 122, 123, 127, 128, 207, 211, 214, or 219.
103021 In certain embodiments, the third polypeptide comprises the
amino acid sequence of
X -.LX2 SX3X4X5LQX8ESVFQS GAHAYRI PAL LYL PGQQSLLAFAEQRX7 SX8X9DEHAEL
IVX_LoRRG
DYDAX THQVQWX12AQEVVAQA.X13LX14GHRSMNPCPLYDX15QT GTL FL FFIAI PX16X17VTEX
i8QQLQTRANVIRLX19X20VT S TDHGRTWSSPRDLTDAAI GPX21YREWST FAVGDGHX22LQLHDX
23X24RSLVVPAYAYRKLHPX25X26X27P IPSAFX2aFLSHDHGRTWARGHFVX29QDTX3oECQVAEV
X311GEQRVV1LNARSX32X33X34X35RX364A4SX37NX38GLDFQX39X4o0X41VKKLX42EPPPX43G
X44QGSVI S FPS PRS GPGSPAQX45LLYTHPTHX46X47QRADLGAYLNPRPPAPEAWSEPX48LLAK
GSX49AYS DLQSMGT GPDGS PL FGX5oLYEANDYEE I X5iFX52MFTLKQAFPAEYL PQX53DKTHT C
PPCPAPELLGGPSVFLFPPKPKLYILMI SRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNS TYRVVSVLTVLHQDWLNGKEYKGKVSNKALPAP EKT SK_AKGQPRE PQVY TLPPS
REEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKT T PPVLDSDGS FFLTSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYT QKSLS LS PGK (SEQ ID NO: 240),
wherein Xi is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr,
Val, or not
present, X2 is Ala or Lys, X3 is Asn or Leu, X4 is Pro or His, X5 is Phe, Trp,
Tyr or Val, X6 is Lys
or Asp, X7 is Ala or Arg, X8 is Lys, Arg, or Glu, X9 is Lys, Ala, Arg, or Glu,
Xio is Leu or Met,
Xi i is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, X12 is Gln or His, X13 is
Arg or Lys, X14 is Asp or
Pro, X15 is Ala, Glu or Lys, X16 is Gly or Asp, X17 is Gln or His, Xis is Gln,
Arg, or Lys, X19 is
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Ala, Cys, Ile, Ser, Val, or Leu, X20 is Gln, Leu, Glu, Phe, His, Ile, Leu, or
Tyr, X21 is Ala or Val,
X22 is Cys or Gly, X23 is Arg or Pro, X24 is Ala or Gly, X25 is Arg, Ile, or
Lys, X26 is Gln or Pro,
X27 is Arg or Pro, X28 is Ala, Cys, Leu, or Val, X29 is Ala, Cys, Asn, Ser, or
Thr, X30 is Leu, Ala,
or Val, X31 is Glu or Pro, X32 is His or Pro, X33 is Leu, Asp, Asn, or Tyr,
X34 is Arg, Ala, Asp,
Leu, Gln, or Tyr, X35 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Gln, Arg, Ser, Val, Trp,
or Tyr, X36 is Val, Ile, or Lys, X37 is Thr or Ala, X38 is Asp or Gly, X39 is
Glu, Lys, or Pro, X40 is
Ser or Cys, X41 is Leu, Asp, Phe, Gln, or Thr, X42 is Val or Phe, X43 is Gln,
Ala, His, Phe, Pro,
Ser, or Thr, X44 is Cys or Val, X45 is Trp or Arg, X46 is Ser, Arg, Ala, Asp,
Glu, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, X47 is Trp, Lys,
Ala, Asp, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, X48 is Lys
or Val, X49 is Ala,
Cys, Ser, or Val, X50 is Cys, Leu, or Val, X51 is Val or Arg, X52 is Leu, Gln,
His, Ile, Lys, or Ser,
and X53 is GGGGS (SEQ ID NO: 121), GGGGSGGGGS (SEQ ID NO: 90), or EPKSS (SEQ
ID
NO: 91), and the sialidase comprises at least one mutation relative to wild-
type human Neu2
(SEQ ID NO: 1).
103031 In certain embodiments, the third polypeptide comprises the amino
acid sequence of
X iASLPX2LQX3E SVFQS GAHAYRI PALLYL PGQQS LLAFAEQRX 4 SKKDEHAEL IVLRRGDYDAX
5 THQVQWQAQEVVAQARL DGHRSMNPCPLYDX 6QT GIL FL FFIAI PGQVTEQQQLQTRANVTRLC
X7VTSTDHGRTWSSPRDL TDAAIGPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPX8Q
RP I PSAFC FL SHDHGRTWARGHEVAQDTLECQVAEVET GEQRVVTLNARSHLRX9RVQAQS TNDG
LDFQESQLVKKLVEPPPX10GCQGSVI S FPS PRS GPGS PAQWL LYTHP THX iiXi2QRADL GAYLN
PRPPAPEAWSEPVLLAKGSX13AYSDLQSMGTGPDGSPLFGCLYEANDYEE I X14 FX15METLKQA
FPAEYLPQX16DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP TENT I SK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVNGFYPSDIAVEWESNGQPENNYKT TPPVLDSDG
S FEL T SKL TVDKSRWQQGNVESCSVMHEALHNHYTQNS L SLS PGK (SEQ ID NO: 241),
wherein Xi is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr,
Val, or not
present, X2 is Phe, Trp, Tyr or Val, X3 is Lys or Asp, X4 is Arg or Ala, X5 is
Pro, Asn, Asp, His,
Glu, Gly, Ser or Thr, X6 is Ala, Glu, or Lys, X7 is Gln, Leu, Glu, Phe, His,
Ile, Leu, or Tyr, Xg is
Arg, Ile, or Lys, X9 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln,
Arg, Ser, Val, Trp,
or Tyr, X10 is Gln, Ala, His, Phe, Pro, Ser, or Thr, XII is Ser, Arg, Ala,
Asp, Glu, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, X12 is Trp, Lys,
Ala, Asp, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, X13 is
Ala, Cys, Ser, or Val,
X14 1 s Val or Arg, Xi5 is Leu, Gln, His, Ile, Lys, or Ser, and X16 is GGGGS
(SEQ ID NO: 121),
GGGGSGGGGS (SEQ ID NO. 90), or EPKSS (SEQ ID NO: 91), and the sialidase
comprises at
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least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1). In certain
embodiments,
X1 is Ala, Asp, Met, or not present, X2 is Tyr or Val, X3 is Lys or Asp, X4 is
Arg or Ala, X5 is
Pro, Asn, Gly, Ser or Thr, X6 is Ala or Glu, X7 is Gln or Tyr, X8 is Ile or
Lys, X9 is Ala or Thr,
Xio is Gln, Ala, or Thr, Xii is Ser, Arg, or Ala, X12 is Trp, Lys, or Arg, X13
is Ala or Cys, X14 is
Val or Arg, and X15 is Leu or Ile.
[0304] In certain embodiments, the first polypeptide comprises SEQ ID NO: 65,
the second
polypeptide comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ
ID NO: 67. In
certain embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 68. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 69. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 70. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 71. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 72. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 73. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 78. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 81. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 82. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 83. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 84. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 85. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 86. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
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comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 87. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 95. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 96. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 98. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 99. In
certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 101.
In certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 102.
In certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 106.
In certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 112.
In certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 127.
In certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 66, and the third polypeptide comprises SEQ ID NO: 128.
In certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 104, and the third polypeptide comprises SEQ ID NO: 108.
In certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 124, and the third polypeptide comprises SEQ ID NO: 122.
In certain
embodiments, the first polypeptide comprises SEQ ID NO: 65, the second
polypeptide
comprises SEQ ID NO: 124, and the third polypeptide comprises SEQ ID NO: 123.
103051 In certain embodiments, the first polypeptide comprises SEQ ID NO: 205,
the second
polypeptide comprises SEQ ID NO: 206, and the third polypeptide comprises SEQ
ID NO: 207.
In certain embodiments, the first polypeptide comprises SEQ ID NO: 205, the
second
polypeptide comprises SEQ ID NO: 206, and the third polypeptide comprises SEQ
ID NO: 211.
In certain embodiments, the first polypeptide comprises SEQ ID NO: 205, the
second
polypeptide comprises SEQ ID NO: 213, and the third polypeptide comprises SEQ
ID NO: 214.
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In certain embodiments, the first polypeptide comprises SEQ ID NO: 205, the
second
polypeptide comprises SEQ ID NO: 213, and the third polypeptide comprises SEQ
ID NO: 219.
103061 In certain embodiments, the antibody conjugate comprises a
first polypeptide
comprising a first sialidase, a first immunoglobulin Fc domain, and a first
single chain variable
fragment (scFv) (it is also understood that the scFv may be replaced by a
first polypeptide chain
of an immunoglobulin antigen binding fragment, e.g., Fab fragment); and a
second polypeptide
comprising a second sialidase, a second immunoglobulin Fc domain, and a second
single chain
variable fragment (scFv) (it is also understood that the scFv may be replaced
by a second
polypeptide chain of an immunoglobulin antigen binding fragment, e.g., Fab
fragment). An
example of this embodiment is shown in FIGURE 6C (in the construct depicted in
FIGURE 6C
it is understood that an scFv, when present, may be replaced with a Fab
fragment, or a Fab
fragment, when present, may be replaced with an scFv). The first and second
polypeptides can
be covalently linked together. The covalent linkages can be disulfide bonds.
In certain
embodiments, the first scFv defines a first anti-PD-Li antigen-binding site,
and the second scFv
defines a second anti-PD-Li antigen-binding site. In certain embodiments, the
first polypeptide
comprises the first sialidase, the first immunoglobulin Fc domain, and the
first scFv in an N- to
C-terminal orientation. In certain embodiments, the first polypeptide
comprises the first scFv,
the first immunoglobulin Fe domain, and the first sialidase in an N- to C-
terminal orientation. In
certain embodiments, the second polypeptide comprises the second sialidase,
the second
immunoglobulin Fc domain, and the second scFv in an N- to C-terminal
orientation. In certain
embodiments, the second polypeptide comprises the second scFv, the second
immunoglobulin
Fc domain, and the second sialidase in an N- to C-terminal orientation.
103071 In certain embodiments, the antibody conjugate comprises: a
first polypeptide
comprising an immunoglobulin light chain; a second polypeptide comprising an
immunoglobulin heavy chain and a single chain variable fragment (scFv) (it is
also understood
that the scFv may be replaced by a first polypeptide chain of an
immunoglobulin antigen binding
fragment, e.g., Fab fragment); and a third polypeptide comprising an
immunoglobulin Fc domain
and a sialidase. It is also understood that the immunoglobulin light chain and
the
immunoglobulin heavy chain variable region may be swapped. An example of this
embodiment
is shown in FIGURE 6D The first and second polypeptides can be covalently
linked together
and the second and third polypeptides can be covalently linked together. The
covalent linkages
can be disulfide bonds. In certain embodiments, the first polypeptide and the
second polypeptide
together define a first anti-PD-Li antigen-binding site (i.e., the
immunoglobulin light chain and
immunoglobulin heavy chain together define a first anti-PD-Li antigen-binding
site). In certain
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embodiments, the scFy defines a second anti-PD-Li antigen-binding site. In
certain
embodiments, the second polypeptide comprises the immunoglobulin heavy chain
and the scFy
in an N- to C-terminal orientation, or the scFy and the immunoglobulin heavy
chain in an N- to
C-terminal orientation. In certain embodiments, the third polypeptide
comprises the sialidase
and the immunoglobulin Fc domain in an N- to C-terminal orientation, or the
sialidase and the
immunoglobulin Fc domain in an N- to C-terminal orientation.
103081 In certain embodiments, the antibody conjugate comprises a
first polypeptide
comprising an immunoglobulin light chain; a second polypeptide comprising a
first sialidase, a
first immunoglobulin Fc domain, and a first immunoglobulin heavy chain
variable region; a third
polypeptide comprising a second sialidase, a second immunoglobulin Fc domain,
and a second
immunoglobulin heavy chain variable region; and a fourth polypeptide
comprising a second
immunoglobulin light chain. It is also understood that an immunoglobulin light
chain may be
replaced by an immunoglobulin heavy chain variable region and an
immunoglobulin heavy
chain variable region may be replaced by an immunoglobulin light chain (e.g.,
the antibody
conjugate may comprise a first polypeptide comprising an immunoglobulin heavy
chain variable
region; a second polypeptide comprising a first sialidase, a first
immunoglobulin Fc domain, and
a first immunoglobulin light chain; a third polypeptide comprising a second
sialidase, a second
immunoglobulin Fc domain, and a second immunoglobulin light chain, and a
fourth polypeptide
comprising a second immunoglobulin heavy chain variable region). An example of
this
embodiment is shown in FIGURE 6E. The second and third polypeptides can be
covalently
linked together. The covalent linkages can be disulfide bonds. In certain
embodiments, the first
and second polypeptides defines a first anti-PD-1 antigen-binding site, and
the third and fourth
polypeptides defines a second anti-PD-1 antigen-binding site. In certain
embodiments, the
second polypeptide comprises the first sialidase, the first immunoglobulin Fc
domain, and the
first immunoglobulin heavy chain variable region in an N- to C-terminal
orientation. In certain
embodiments, the third polypeptide comprises the second sialidase, the second
immunoglobulin
Fc domain, and the second immunoglobulin heavy chain variable region in an N-
to C-terminal
orientation.
103091 In certain embodiments, the antibody conjugate has a molecular weight
from about 135
kDa to about 165 kDa, e.g., about 140 kDa In other embodiments, the antibody
conjugate has a
molecular weight from about 215 kDa to about 245 kDa, e.g., about 230 kDa.
103101 In certain embodiments, the antibody conjugate comprises two
polypeptides that each
comprise an immunoglobulin Fc domain, and the first polypeptide has either a
"knob- mutation,
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e.g., T366Y, or a "hole- mutation, e.g, Y407T, for heterodimerization with the
second
polypeptide, and the second polypeptide has either a respective "knob"
mutation, e.g., T366Y, or
a "hole" mutation, e.g., Y407T, for heterodimerization with the first
polypeptide (residue
numbers according to EU numbering, Kabat, E.A., et al. (1991) supra). For
example, in certain
embodiments, the antibody comprises two polypeptides that each comprise an
immunoglobulin
Fc domain derived from human IgG1 Fc domain, and the first polypeptide
comprises a Y407T
mutation (e.g., the first polypeptide comprises SEQ ID NO: 32 or SEQ ID NO:
92), and the
second polypeptide comprises a T366Y mutation (e.g., the second polypeptide
comprises SEQ
ID NO: 33 or SEQ ID NO: 93).
103111 In certain embodiments, the antibody conjugate comprises an
immunoglobulin Fc
domain that is modified to prevent to glycosylation of the Fc domain. For
example, in certain
embodiments, the immunoglobulin Fc domain is derived from a human IgG1 Fc
domain and
comprises a mutation at position N297, for example, an N297A or N297G mutation
(residue
numbers according to EU numbering, Kabat, E.A., et al., supra). For example,
in certain
embodiments, the antibody conjugate comprises SEQ ID NO: 222, SEQ ID NO: 225,
or SEQ ID
NO: 226.
103121 As used herein, the term "multispecific antibody" is understood to mean
an antibody
that specifically binds to at least two different antigens, i.e., an antibody
that comprises at least
two antigen-binding sites that bind to at least two different antigens. As
used herein, the term
"bispecific antibody" is understood to mean an antibody that specifically
binds to two different
antigens, i.e., an antibody that comprises two antigen-binding sites each of
which bind to
separate and distinct antigens. In other words, a first binding site binds a
first antigen and a
second binding site binds a second, different antigen. A multispecific or
bispecific antibody
may, for example, be a human or humanized antibody, and/or be a full length
antibody or an
antibody fragment (e.g., a F(ab')2 bispecific antibody).
103131 The present invention encompasses antibody conjugates comprising
antibody
fragments, which may be generated by traditional means, such as enzymatic
digestion, or by
recombinant techniques. For a review of certain antibody fragments, see Hudson
et al. (2003)
supra.
103141 In certain embodiments, the antibody conjugate or fusion protein can be
covalently or
non-covalently associated with a biological modifier, wherein the biological
modifier can be
used to enhance the solubility of the antibody, increase binding specificity,
decrease
immunogenicity or toxicity or modify the pharmacokinetic profile of the
antibody. For example,
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the biological modifier can be used to increase the molecular weight of the
antibody to increase
its circulating half-life.
103151 It is contemplated that the antibody conjugate or fusion protein may be
covalently
bound to one or more (for example, 2, 3, 4, 5, 6, 8, 9, 10 or more) biological
modifiers that may
comprise linear or branched polymers. Exemplary biological modifiers may
include, for
example, a variety of polymers, such as those described in U.S. Patent No.
7,842,789.
Particularly useful are polyalkylene ethers such as polyethylene glycol (PEG)
and derivatives
thereof (for example, alkoxy polyethylene glycol, for example,
methoxypolyethylene glycol,
ethoxypolyethylene glycol and the like); block copolymers of polyoxyethylene
and
polyoxypropylene (Pluronics); polymethacrylates; carbomers; and branched or
unbranched
polysaccharides which comprise the saccharide monomers such as D-mannose, D-
and L-
galactose, fucose, fructose, D-xylose, L-arabinose, and D-glucuronic acid.
103161 In other embodiments, the biological modifier can be a hydrophilic
polyvinyl polymer
such as polyvinyl alcohol and polyvinylpyrrolidone (PVP)-type polymers. The
biological
modifier can be a functionalized polyvinylpyrrolidone, for example, carboxy or
amine
functionalized on one (or both) ends of the polymer (as available from
PolymerSource).
Alternatively, the biological modifier can include Poly N-(2-
hydroxypropyl)methacrylamide
(HPMA), or functionalized HPMA (amine, carboxy, etc.), Poly(N-
isopropylacrylamide) or
functionalized poly(N-isopropylacrylamide). Alternatively, the biological
modifier can include
Poly N-(2-hydroxypropyl)methacrylamide (HPMA), or functionalized HPMA (amine,
carboxy,
etc.), Poly(N-isopropylacrylamide) or functionalized poly(N-
isopropylacrylamide). The
modifier prior to conjugation need not be, but preferably is, water soluble,
but the final conjugate
should be water soluble.
103171 In general, the biological modifier may have a molecular weight from
about 2 kDa to
about 5 kDa, from about 2 kDa to about 10 kDa, from about 2 kDa to about 20
kDa, from about
2 kDa to about 30 kDa, from about 2 kDa to about 40 kDa, from about 2 kDa to
about 50 kDa,
from about 2 kDa to about 60 kDa, from about 2 kDa to about 70 kDa, from about
2 kDa to
about 80 kDa, from about 2 kDa to about 90 kDa, from about 2 kDa to about 100
kDa, from
about 2 kDa to about 150 kDa, from about 5 kDa to about 10 kDa, from about 5
kDa to about 20
kDa, from about 5 kDa to about 30 kDa, from about 5 kDa to about 40 kDa, from
about 5 kDa to
about 50 kDa, from about 5 kDa to about 60 kDa, from about 5 kDa to about 70
kDa, from about
5 kDa to about 80 kDa, from about 5 kDa to about 90 kDa, from about 5 kDa to
about 100 kDa,
from about 5 kDa to about 150 kDa, from about 10 kDa to about 20 kDa, from
about 10 kDa to
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about 30 kDa, from about 10 kDa to about 40 kDa, from about 10 kDa to about 50
kDa, from
about 10 kDa to about 60 kDa, from about 10 kDa to about 70 kDa, from about 10
kDa to about
80 kDa, from about 10 kDa to about 90 kDa, from about 10 kDa to about 100 kDa,
from about
kDa to about 150 kDa, from about 20 kDa to about 30 kDa, from about 20 kDa to
about 40
5 kDa, from about 20 kDa to about 50 kDa, from about 20 kDa to about 60
kDa, from about 20
kDa to about 70 kDa, from about 20 kDa to about 80 kDa, from about 20 kDa to
about 90 kDa,
from about 20 kDa to about 100 kDa, from about 20 kDa to about 150 kDa, from
about 30 kDa
to about 40 kDa, from about 30 kDa to about 50 kDa, from about 30 kDa to about
60 kDa, from
about 30 kDa to about 70 kDa, from about 30 kDa to about 80 kDa, from about 30
kDa to about
10 90 kDa, from about 30 kDa to about 100 kDa, from about 30 kDa to about
150 kDa, from about
40 kDa to about 50 kDa, from about 40 kDa to about 60 kDa, from about 40 kDa
to about 70
kDa, from about 40 kDa to about 80 kDa, from about 40 kDa to about 90 kDa,
from about 40
kDa to about 100 kDa, from about 40 kDa to about 150 kDa, from about 50 kDa to
about 60
kDa, from about 50 kDa to about 70 kDa, from about 50 kDa to about 80 kDa,
from about 50
kDa to about 90 kDa, from about 50 kDa to about 100 kDa, from about 50 kDa to
about 150
kDa, from about 60 kDa to about 70 kDa, from about 60 kDa to about 80 kDa,
from about 60
kDa to about 90 kDa, from about 60 kDa to about 100 kDa, from about 60 kDa to
about 150
kDa, from about 70 kDa to about 80 kDa, from about 70 kDa to about 90 kDa,
from about 70
kDa to about 100 kDa, from about 70 kDa to about 150 kDa, from about 80 kDa to
about 90
kDa, from about 80 kDa to about 100 kDa, from about 80 kDa to about 150 kDa,
from about 90
klla to about 100 klla, from about 90 kDa to about 150 kDa, or from about 100
kDa to about
150 kDa.
103181 It is contemplated that the antibody conjugate or fusion protein is
attached to about 10
or fewer polymer molecules (e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1), each polymer
molecule having a
molecular weight of at least about 20,000 D, or at least about 30,000 D, or at
least about
40,000 D.
103191 Although a variety of polymers can be used as biological modifiers, it
is contemplated
that the antibody conjugates or fusion proteins described herein may be
attached to polyethylene
glycol (PEG) polymers. In one embodiment, the antibody conjugate or fusion
protein described
herein is covalently attached to at least one PEG having an actual MW of at
least about 20,000
D. In another embodiment, the antibody conjugate or fusion protein described
herein is
covalently attached to at least one PEG having an actual MW of at least about
30,000 D. In
another embodiment, the antibody conjugate or fusion protein described herein
is covalently
attached to at least one PEG having an actual MW of at least about 40,000 D.
In certain
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embodiments, the PEG is methoxyPEG(5000)-succinimidylpropionate (mPEG-SPA),
methoxyPEG(5000)-succinimidylsuccinate (mPEG-SS). Such PEGS are commercially
available
from Nektar Therapeutics or SunBiowest.
103201 Attachment sites on an antibody conjugate or fusion protein for a
biological modifier
include the N-terminal amino group and epsilon amino groups found on lysine
residues, as well
as other amino, imino, carboxyl, sulfhydryl, hydroxyl or other hydrophilic
groups. The polymer
may be covalently bonded directly to the antibody conjugate or fusion protein
with or without
the known use of a multifunctional (ordinarily bifunctional) cros slinking
agent using chemistries
and used in the art. For example, sulfhydryl groups can be derivatized by
coupling to
maleimido-substituted PEG (e.g., alkoxy-PEG amine plus sulfosuccinimidyl 4-(N-
maleimidomethyl)cyclohexane-1-carboxylate), or PEG-maleimide commercially
available from
Shearwater Polymers, Inc., Huntsville, Ala.).
II. Methods of Making an Antibody, Fusion Protein, or Antibody
Conjugate
103211 Methods for producing antibodies, fusion proteins, or antibody
conjugates, e.g., those
disclosed herein, are known in the art For example, DNA molecules encoding
light chain
variable regions and/or heavy chain variable regions can be synthesized
chemically or by
recombinant DNA methodologies. For example, the sequences of the antibodies
can be cloned
from hybridomas by conventional hybridization techniques or polymerase chain
reaction (PCR)
techniques, using the appropriate synthetic nucleic acid primers. The
resulting DNA molecules
encoding the variable legions of interest can be ligated to other appropriate
nucleotide
sequences, including, for example, constant region coding sequences, and
expression control
sequences, to produce conventional gene expression constructs (i.e.,
expression vectors)
encoding the desired antibodies. Production of defined gene constructs is
within routine skill in
the art.
[0322] Nucleic acids encoding desired antibodies, fusion proteins, and/or
antibody conjugates
can be incorporated (ligated) into expression vectors, which can be introduced
into host cells
through conventional transfection or transformation techniques. Exemplary host
cells are E. col?
cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293)
cells, HeLa
cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human
hepatocellular
carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise
produce IgG protein.
Transformed host cells can be grown under conditions that permit the host
cells to express the
genes that encode the immunoglobulin light and/or heavy chain variable
regions.
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103231 Specific expression and purification conditions will vary depending
upon the
expression system employed. For example, if a gene is to be expressed in E.
coil, it is first
cloned into an expression vector by positioning the engineered gene downstream
from a suitable
bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence. The
expressed protein
may be secreted. The expressed protein may accumulate in refractile or
inclusion bodies, which
can be harvested after disruption of the cells by French press or sonication.
The refractile bodies
then are solubilized, and the protein may be refolded and/or cleaved by
methods known in the
art.
103241 If the engineered gene is to be expressed in eukaryotic host cells,
e.g., CHO cells, it is
first inserted into an expression vector containing a suitable eukaryotic
promoter, a secretion
signal, a poly A sequence, and a stop codon. Optionally, the vector or gene
construct may
contain enhancers and introns. In embodiments involving antibodies or fusion
proteins
comprising an antibody or portion thereof, the expression vector optionally
contains sequences
encoding all or part of a constant region, enabling an entire, or a part of, a
heavy or light chain to
be expressed. The gene construct can be introduced into eukaryotic host cells
using
conventional techniques.
103251 In certain embodiments, the host cells express an antibody, fusion
protein and/or
antibody conjugate comprising a sialidase and VL or VH fragments, VL-VH
heterodimers, VH-VL
or VL-Vii single chain polypeptides, complete heavy or light immunoglobulin
chains, or portions
thereof, each of which may be attached to a moiety having another function
(e.g., cytotoxicity).
In some embodiments involving antibodies, fusion proteins and/or antibody
conjugates, a host
cell is transfected with a single vector expressing a polypeptide expressing a
sialidase and an
entire, or part of, a heavy chain (e.g., a heavy chain variable region) or a
sialidase and a light
chain (e.g., a light chain variable region), or a polypeptide expressing an
entire, or part of, a
heavy chain (e.g., a heavy chain variable region) or a light chain (e.g., a
light chain variable
region). In some embodiments, a host cell is transfected with a single vector
encoding (a) a
polypeptide comprising a heavy chain variable region and a polypeptide
comprising a light chain
variable region, or (b) an entire immunoglobulin heavy chain and an entire
immunoglobulin light
chain, wherein in (a) or in (b), the polypeptide may also comprise a
sialidase. In some
embodiments, a host cell is co-transfected with more than one expression
vector (e.g., one
expression vector expressing a polypeptide comprising an entire, or part of, a
heavy chain or
heavy chain variable region, optionally comprising a sialidase fused thereto,
and another
expression vector expressing a polypeptide comprising an entire, or part of, a
light chain or light
chain variable region, optionally comprising a sialidase fused thereto).
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103261 A polypeptide comprising an antibody or a fusion protein, e.g., an
antibody or a fusion
protein comprising an immunoglobulin heavy chain variable region and/or light
chain variable
region, can be produced by growing (culturing) a host cell transfected with an
expression vector
encoding such a variable region, under conditions that permit expression of
the polypeptide.
Following expression, the polypeptide can be harvested and purified or
isolated using techniques
known in the art, e.g., affinity tags such as glutathione-S-transferase (GST)
or histidine tags.
103271 In certain embodiments, an antibody, fusion protein, and/or antibody
conjugate can be
produced by growing (culturing) a host cell transfected with: (a) an
expression vector that
encodes a complete or partial immunoglobulin heavy chain, and a separate
expression vector that
encodes a complete or partial immunoglobulin light chain; or (b) a single
expression vector that
encodes both chains (e.g., complete or partial heavy and light chains), under
conditions that
permit expression of both chains. In embodiments in which a fusion protein
and/or antibody
conjugate is produced, the sialidase is fused to one or more of the chains.
The intact antibody,
fusion protein, and/or antibody conjugate can be harvested and purified or
isolated using
techniques known in the art, e.g., Protein A, Protein G, affinity tags such as
glutathione-S-
transferase (GST) or histidine tags. It is within ordinary skill in the art to
express the heavy
chain and the light chain from a single expression vector or from two separate
expression
vectors.
103281 In certain embodiments, in order to express a protein, e.g.,
an antibody and/or a
fusion protein, as a secreted protein, a native N-terminal signal sequence of
the protein is
replaced, e.g., with MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28). In certain
embodiments, to express a protein, e.g., an antibody and/or a fusion protein,
as a secreted
protein, an 1N-terminal signal sequence, e.g., MDMRVPAQLLGLLLLWLPGARC (SEQ ID
NO:
28), is added. Additional exemplary N-terminal signal sequences include signal
sequences from
interleukin-2, CD-5, IgG kappa light chain, trypsinogen, serum albumin, and
prolactin. In
certain embodiments, in order to express a protein, e.g., an antibody and/or a
fusion protein, as a
secreted protein, a C terminal lysosomal signal motif, e.g., YGTL (SEQ ID NO:
29) is removed.
103291 Methods for reducing or eliminating the antigenicity of antibodies and
antibody
fragments are known in the art. When the antibodies are to be administered to
a human, the
antibodies preferably are "humanized" to reduce or eliminate antigenicity in
humans.
Preferably, each humanized antibody has the same or substantially the same
affinity for the
antigen as the non-humanized mouse antibody from which it was derived.
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[0330] In one humanization approach, chimeric proteins are created in which
mouse
immunoglobulin constant regions are replaced with human immunoglobulin
constant regions.
See, e.g., Morrison et at., 1984, PROC. NAT. ACAD. SO. 81:6851-6855, Neuberger
et at., 1984,
NATURE 312:604-608; U.S. Patent Nos. 6,893,625 (Robinson); 5,500,362
(Robinson); and
4,816,567 (Cabilly).
[0331] In an approach known as CDR grafting, the CDRs of the light and heavy
chain variable
regions are grafted into frameworks from another species. For example, murine
CDRs can be
grafted into human FRs. In some embodiments, the CDRs of the light and heavy
chain variable
regions of an antibody are grafted into human FRs or consensus human FRs. To
create
consensus human FRs, FRs from several human heavy chain or light chain amino
acid sequences
are aligned to identify a consensus amino acid sequence. CDR grafting is
described in U.S.
Patent Nos. 7,022,500 (Queen), 6,982,321 (Winter); 6,180,370 (Queen);
6,054,297 (Carter);
5,693,762 (Queen); 5,859,205 (Adair); 5,693,761 (Queen); 5,565,332
(Hoogenboom); 5,585,089
(Queen); 5,530,101 (Queen); Jones etal. (1986) NATURE 321: 522-525; Riechmann
et at. (1988)
NATURE 332: 323-327; Verhoeyen et al. (1988) SCIENCE 239: 1534-1536; and
Winter (1998)
FEBS LETT 430: 92-94.
[0332] In an approach called "SUPERHUMANIZATION¨," human CDR sequences are
chosen from human germline genes, based on the structural similarity of the
human CDRs to
those of the mouse antibody to be humanized. See, e.g., U.S. Patent No.
6,881,557 (Foote); and
Tan etal., 2002, J. ImivruNoL. 169:1119-1125.
[0333] Other methods to reduce immunogenicity include "reshaping," -
hyperchimerization,"
and "veneering/resurfacing." See, e.g., Vaswami et at., 1998, ANNALS OF
ALLERGY, ASTHMA, &
IMMUNOL. 81:105; Roguska et al., 1996, PROT. ENGINEER 9:895-904; and U.S.
Patent No.
6,072,035 (Hardman). In the veneering/resurfacing approach, the surface
accessible amino acid
residues in the murine antibody are replaced by amino acid residues more
frequently found at the
same positions in a human antibody. This type of antibody resurfacing is
described, e.g-., in U.S.
Patent No. 5,639,641 (Pedersen).
[0334] Another approach for converting a mouse antibody into a form suitable
for medical use
in humans is known as ACTIVMAW' technology (Vaccinex, Inc., Rochester, NY),
which
involves a vaccinia virus-based vector to express antibodies in mammalian
cells. High levels of
combinatorial diversity of IgG heavy and light chains can be produced. See,
e.g., U.S. Patent
Nos. 6,706,477 (Zauderer); 6,800,442 (Zauderer); and 6,872,518 (Zauderer).
Another approach
for converting a mouse antibody into a form suitable for use in humans is
technology practiced
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commercially by KaloBios Pharmaceuticals, Inc. (Palo Alto, CA). This
technology involves the
use of a proprietary human "acceptor" library to produce an "epitope focused"
library for
antibody selection. Another approach for modifying a mouse antibody into a
form suitable for
medical use in humans is HUMAN ENGiNEERINGTM technology, which is practiced
commercially by XOMA (US) LLC. See, e.g., International (PCT) Publication No.
WO
93/11794 and U.S. Patent Nos. 5,766,886 (Studnicka); 5,770,196 (Studnicka);
5,821,123
(Studnicka); and 5,869,619 (Studnicka).
103351 Any suitable approach, including any of the above approaches, can be
used to reduce or
eliminate human immunogenicity of an antibody.
103361 In addition, it is possible to create fully human antibodies in mice.
Fully human mAbs
lacking any non-human sequences can be prepared from human immunoglobulin
transgenic
mice by techniques referenced in, e.g., Lonberg et al., NATURE 368:856-859,
1994; Fishwild et
at., NATURE BIOTECHNOLOGY 14:845-851, 1996; and Mendez et at., NATURE GENETICS
15:146-156, 1997. Fully human monoclonal antibodies can also be prepared and
optimized from
phage display libraries by techniques referenced in, e.g., Knappik et at., J.
MOL. BIOL. 296:57-
86, 2000; and Krebs et at., J. IM1VIENOL. METH. 254:67-84 2001).
103371 The present invention encompasses antibody fragments, or fusion
proteins comprising
antibody fragments, which may be generated by traditional means, such as
enzymatic digestion,
or by recombinant techniques. For a review of certain antibody fragments, see
Hudson et at.
(2003) NAT. MED. 9.129-134.
103381 Various techniques have been developed for the production of antibody
fragments.
Traditionally, these fragments were derived via proteolytic digestion of
intact antibodies (see,
e.g.,Morimoto et at. (1992) JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS
24:107-
117; and Brennan et at. (1985) SCIENCE 229:81). However, these fragments can
now be
produced directly by recombinant host cells. Fab, Fv and ScFy antibody
fragments can all be
expressed in and secreted from E. coil, thus allowing the facile production of
large amounts of
these fragments. Antibody fragments can be isolated from the antibody phage
libraries.
Alternatively, Fab'-SH fragments can be directly recovered from E. colt and
chemically coupled
to form F(ab')2 fragments (Carter etal. (1992) BIO/TECHNOLOGY 10:163-167).
According to
another approach, F(ab')2 fragments can be isolated directly from recombinant
host cell culture.
Fab and F(ab')2 fragments with increased in vivo half-life comprising salvage
receptor binding
epitope residues are described in U.S. Patent No. 5,869,046. Other techniques
for the production
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of antibody fragments will be apparent to the skilled practitioner. In certain
embodiments, an
antibody is a single chain Fv fragment (scFv). See U.S. Patent Nos. 5,571,894
and 5,587,458.
103391 Methods for making bispecific antibodies are known in the art. See
Milstein and
Cuello (1983) NATURE 305:537, International (PCT) Publication No. W093/08829,
and
Traunecker et al. (1991) ElV1130 J., 10:3655. For further details of
generating bispecific
antibodies see, for example, Suresh et al. (1986) METHODS ENZYMOL. 121:210.
Bispecific
antibodies include cross-linked or "heteroconjugate" or "heterodimer"
antibodies. For example,
one of the antibodies in the heterodimer can be coupled to avidin, the other
to biotin.
Heterodimer antibodies may be made using any convenient cross-linking method.
Suitable
cross-linking agents are well known in the art, and are disclosed in U.S.
Patent No. 4,676,980,
along with a number of cross-linking techniques.
103401 Examples of heterodimeric or asymmetric IgG-like molecules include but
are not
limited to those obtained with the following technologies or using the
following formats:
Triomab/Quadroma, Knobs-into-Holes, CrossMabs, electrostatically-matched
antibodies, LUZ-
Y, Strand Exchange Engineered Domain body, Biclonic and DuoBody.
103411 Advantages of using antibody fragments (e.g., F(ab) and F(ab')2
fragments) include the
elimination of non-specific binding between Fc portions of antibodies and Fc
receptors on cells
(such as macrophages, dendritic cells, neutrophils, NK cells and B cells). In
addition, they may
be able to penetrate tissues more efficiently due to their smaller size.
103421 Heterodimeric antibodies, or asymmetric antibodies, allow for greater
flexibility and
new formats for attaching a variety of drugs to the antibody arms. One of the
general formats for
creating a heterodimeric antibody is the -knobs-into-holes" format. This
format is specific to the
heavy chain part of the constant region in antibodies. The "knobs" part is
engineered by
replacing a small amino acid with a larger one, which fits into a "hole",
which is engineered by
replacing a large amino acid with a smaller one. What connects the "knobs" to
the "holes" are
the disulfide bonds between each chain. The "knobs-into-holes" shape
facilitates antibody
dependent cell mediated cytotoxicity. Single chain variable fragments (scFv)
are connected to
the variable domain of the heavy and light chain via a short linker peptide.
The linker is rich in
glycine, which gives it more flexibility, and serine/threonine, which gives it
specificity. Two
different scFv fragments can be connected together, via a hinge region, to the
constant domain of
the heavy chain or the constant domain of the light chain. This gives the
antibody bispecificity,
allowing for the binding specificities of two different antigens. The -knobs-
into-holes" format
enhances heterodimer formation but doesn't suppress homodimer formation.
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103431 Several approaches to support heterodimerization have been described,
for example in
International (PCT) Publication Nos. W096/27011, W098/050431, W02007/110205,
W02007/147901, W02009/089004, W02010/129304, W02011/90754, W02011/143545,
W02012/058768, W02013/157954, and W02013/096291, and European Patent
Publication No.
EP1870459. Typically, in the approaches known in the art, the CH3 domain of
the first heavy
chain and the CH3 domain of the second heavy chain are both engineered in a
complementary
manner so that the heavy chain comprising one engineered CH3 domain can no
longer
homodimerize with another heavy chain of the same structure (e.g., a CH3-
engineered first heavy
chain can no longer homodimerize with another CH3-engineered first heavy
chain; and a CH3-
engineered second heavy chain can no longer homodimerize with another CH3-
engineered
second heavy chain). Thereby the heavy chain comprising one engineered CH3
domain is forced
to heterodimerize with another heavy chain comprising the CH3 domain, which is
engineered in
a complementary manner. As a result, the CH3 domain of the first heavy chain
and the CH3
domain of the second heavy chain are engineered in a complementary manner by
amino acid
substitutions, such that the first heavy chain and the second heavy chain are
forced to
heterodimerize, whereas the first heavy chain and the second heavy chain can
no longer
homodimerize (e.g., for steric reasons).
III. Pharmaceutical Compositions
103441 For therapeutic use, an antibody, fusion protein, and/or antibody
conjugate preferably is
combined with a pharmaceutically acceptable carrier. The term
"pharmaceutically acceptable"
as used herein refers to those compounds, materials, compositions, and/or
dosage forms which
are, within the scope of sound medical judgment, suitable for use in contact
with the tissues of
human beings and animals without excessive toxicity, irritation, allergic
response, or other
problem or complication, commensurate with a reasonable benefit/risk ratio.
103451 The term "pharmaceutically acceptable carrier- as used herein refers to
buffers,
carriers, and excipients suitable for use in contact with the tissues of human
beings and animals
without excessive toxicity, irritation, allergic response, or other problem or
complication,
commensurate with a reasonable benefit/risk ratio Pharmaceutically acceptable
carriers include
any of the standard pharmaceutical carriers, such as a phosphate buffered
saline solution, water,
emulsions (e.g., such as an oil/water or water/oil emulsions), and various
types of wetting agents.
The compositions also can include stabilizers and preservatives. For examples
of carriers,
stabilizers and adjuvants, see, e.g., Martin, Remington's Pharmaceutical
Sciences, 15th Ed.,
Mack Publ. Co., Easton, PA [1975]. Pharmaceutically acceptable carriers
include buffers,
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solvents, dispersion media, coatings, isotonic and absorption delaying agents,
and the like, that
are compatible with pharmaceutical administration. The use of such media and
agents for
pharmaceutically active substances is known in the art.
103461 In certain embodiments, a pharmaceutical composition may contain
formulation
materials for modifying, maintaining or preserving, for example, the pH,
osmolarity, viscosity,
clarity, color, isotonicity, odor, sterility, stability, rate of dissolution
or release, adsorption or
penetration of the composition. In such embodiments, suitable formulation
materials include,
but are not limited to, amino acids (such as glycine, glutamine, asparagine,
arginine or lysine);
antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium
hydrogen-sulfite);
buffers (such as borate, bicarbonate, Tris-HC1, citrates, phosphates or other
organic acids);
bulking agents (such as mannitol or glycine); chelating agents (such as
ethylenediamine
tetraacetic acid (EDTA)); complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-
cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; m onosacchari des;
di saccharides; and
other carbohydrates (such as glucose, mannose or dextrins); proteins (such as
serum albumin,
gelatin or immunoglobulins); coloring, flavoring and diluting agents;
emulsifying agents;
hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight
polypeptides, salt-
forming counterions (such as sodium), preservatives (such as benzalkonium
chloride, benzoic
acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben,
propylparaben, chlorhexidine,
sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene
glycol or polyethylene
glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents;
surfactants or wetting
agents (such as pluronics, PEG, sorbitan esters, polysorbates such as
polysorbate 20,
polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal);
stability enhancing agents
(such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal
halides, preferably
sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents;
excipients and/or
pharmaceutical adjuvants (see, Remington 's Pharmaceutical Sciences, 18th ed.
(Mack
Publishing Company, 1990).
103471 In certain embodiments, a pharmaceutical composition may contain
nanoparticles, e.g.,
polymeric nanoparticles, liposomes, or micelles (See Anselmo etal. (2016)
BIOENG. TRANSL
MED. 1: 10-29).
103481 In certain embodiments, a pharmaceutical composition may contain a
sustained- or
controlled-delivery formulation. Techniques for formulating sustained- or
controlled-delivery
means, such as liposome carriers, bio-erodible microparticles or porous beads
and depot
injections, are also known to those skilled in the art. Sustained-release
preparations may include,
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e.g., porous polymeric microparticles or semipermeable polymer matrices in the
form of shaped
articles, e.g., films, or microcapsules. Sustained release matrices may
include polyesters,
hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-
glutamate, poly (2-
hydroxy ethyl-inethacrylate), ethylene vinyl acetate, or poly-D(¨)-3-
hydroxybutyric acid.
Sustained release compositions may also include liposomes that can be prepared
by any of
several methods known in the art.
103491 Pharmaceutical compositions containing an antibody, sialidase fusion
protein, or an
antibody conjugate disclosed herein can be presented in a dosage unit form and
can be prepared
by any suitable method. A pharmaceutical composition should be formulated to
be compatible
with its intended route of administration. Examples of routes of
administration are intravenous
(IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal
and rectal
administration. In certain embodiments, an antibody, sialidase fusion protein,
or an antibody
conjugate disclosed herein is administered by IV infusion. In certain
embodiments, an antibody,
sialidase fusion protein, or an antibody conjugate disclosed herein is
administered by
intratumoral injection. Useful formulations can be prepared by methods known
in the
pharmaceutical art. For example, see Remington 's Pharmaceutical Sciences,
18th ed. (Mack
Publishing Company, 1990). Formulation components suitable for parenteral
administration
include a sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene
glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial
agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or
sodium bisulfite;
chelating agents such as EDTA; buffers such as acetates, citrates or
phosphates; and agents for
the adjustment of tonicity such as sodium chloride or dextrose.
103501 For intravenous administration, suitable carriers include physiological
saline,
bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate
buffered saline
(PBS). The carrier should be stable under the conditions of manufacture and
storage, and should
be preserved against microorganisms. The carrier can be a solvent or
dispersion medium
containing, for example, water, ethanol, polyol (for example, glycerol,
propylene glycol, and
liquid polyethylene glycol), and suitable mixtures thereof
103511 In certain embodiments, a pharmaceutical composition may contain a
stabilizing agent.
In certain embodiments, the stabilizing agent is a cation, such as a divalent
cation. In certain
embodiments, the cation is calcium or magnesium. The cation can be in the form
of a salt, such
as calcium chloride (CaCl2) or magnesium chloride (MgCl2).
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103521 In certain embodiments, the stabilizing agent is present in an amount
from about 0.05
mM to about 5 mM. For example, the stabilizing agent may be present in an
amount of from
about 0.05 mM to about 4 mM, from about 0.05 mM to about 3 mM, from about 0.05
mM to
about 2 mM, from about 0.05 mM to about 1 mM, from about 0.05 mM to about 0.5
mM, from
about 0.5 mM to about 4 mM, from about 0.5 mM to about 3 mM, from about 0.5 mM
to about 2
mM, from about 0.5 mM to about 1 mM, from about 1 mM to about 4 mM, from about
1 mM to
about 3 mM, of from about 1 mM to about 2 mM.
103531 Pharmaceutical formulations preferably are sterile. Sterilization can
be accomplished
by any suitable method, e.g., filtration through sterile filtration membranes.
Where the
composition is lyophilized, filter sterilization can be conducted prior to or
following
lyophilization and reconstitution.
103541 The compositions described herein may be administered locally or
systemically.
Administration will generally be parenteral administration. In a preferred
embodiment, the
pharmaceutical composition is administered subcutaneously and in an even more
preferred
embodiment intravenously. Preparations for parenteral administration include
sterile aqueous or
non-aqueous solutions, suspensions, and emulsions.
103551 Generally, a therapeutically effective amount of active component, for
example, an
antibody, fusion protein, and/or antibody conjugate, is in the range of 0.1
mg/kg to 100 mg/kg,
e.g., 1 mg/kg to 100 mg/kg, 1 mg/kg to 10 mg/kg. The amount administered will
depend on
variables such as the type and extent of disease or indication to be treated,
the overall health of
the patient, the in vivo potency of the antibody, the pharmaceutical
formulation, and the route of
administration. The initial dosage can be increased beyond the upper level in
order to rapidly
achieve the desired blood-level or tissue-level. Alternatively, the initial
dosage can be smaller
than the optimum, and the daily dosage may be progressively increased during
the course of
treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose
escalation study
designed to run from 0.5 mg/kg to 20 mg/kg. Dosing frequency can vary,
depending on factors
such as route of administration, dosage amount, serum half-life of the
antibody, fusion protein,
and/or antibody conjugate, and the disease being treated. Exemplary dosing
frequencies are
once per day, once per week and once every two weeks. A preferred route of
administration is
parenteral, e.g., intravenous infusion. In certain embodiments, an antibody,
fusion protein,
and/or antibody conjugate is lyophilized, and then reconstituted in buffered
saline, at the time of
administration.
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IV. Therapeutic Uses
103561 The compositions and methods disclosed herein can be used to treat
various forms of
cancer in a subject or inhibit cancer growth in a subject. The invention
provides a method of
treating a cancer in a subject. The method comprises administering to the
subject an effective
amount of an anti-PD-Li antibody, a sialidase anti-PD-Li fusion protein,
and/or antibody
conjugate, e.g., an antibody, fusion protein, or antibody conjugate disclosed
herein, either alone
or in a combination with another therapeutic agent to treat the cancer in the
subject. The term
"effective amount" as used herein refers to the amount of an active agent (e.g-
., an antibody,
fusion protein, or antibody conjugate according to the present invention)
sufficient to effect
beneficial or desired results. An effective amount can be administered in one
or more
administrations, applications or dosages and is not intended to be limited to
a particular
formulation or administration route.
103571 As used herein, "treat", "treating" and "treatment" mean the treatment
of a disease in a
subject, e.g., in a human. This includes: (a) inhibiting the disease, i.e.,
arresting its development
and (b) relieving the disease, i.e., causing regression of the disease state.
As used herein, the
terms "subject- and "patient- refer to an organism to be treated by the
methods and compositions
described herein. Such organisms preferably include, but are not limited to,
mammals (e.g.,
murines, simians, equines, bovines, porcines, canines, felines, and the like),
and more preferably
includes humans.
103581 Examples of cancers include solid tumors, soft tissue tumors,
hematopoietic tumors and
metastatic lesions. Examples of hematopoietic tumors include, leukemia, acute
leukemia, acute
lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid
leukemia (AML),
chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), e.g.,
transformed
CLL, diffuse large B-cell lymphomas (DLBCL), follicular lymphoma, hairy cell
leukemia,
myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignant
lymphoma, non-
Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, or Richter's
Syndrome
(Richter's Transformation). Examples of solid tumors include malignancies,
e.g., sarcomas,
adenocarcinomas, and carcinomas, of the various organ systems, such as those
affecting head
and neck (including pharynx), thyroid, lung (small cell or non-small cell lung
carcinoma
(NSCLC)), breast, lymphoid, gastrointestinal (e.g., oral, esophageal, stomach,
liver, pancreas,
small intestine, colon and rectum, anal canal), genitals and genitourinary
tract (e.g., renal,
urothelial, bladder, ovarian, uterine, cervical, endometrial, prostate,
testicular), CNS (e.g., neural
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or glial cells, e.g., neuroblastoma or glioma), or skin (e.g., melanoma and
metastatic Merkel cell
carcinoma (MCC)).
103591 In certain embodiments the cancer is an epithelial cancer, e.g., an
epithelial cancer that
upregulates the expression of sialylated glycans Exemplary epithelial cancers
include, but are
not limited to, endometrial cancer, colon cancer, ovarian cancer, cervical
cancer, vulyar cancer,
uterine cancer or fallopian tube cancer, breast cancer, prostate cancer, lung
cancer, pancreatic
cancer, urinary cancer, bladder cancer, head and neck cancer, oral cancer and
liver cancer.
Epithelial cancers also include carcinomas, for example, acinar carcinoma,
acinous carcinoma,
adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum,
carcinoma of
adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell
carcinoma, carcinoma
basocellulare, basaloid carcinoma, baso squamous cell carcinoma,
bronchioalyeolar carcinoma,
bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma,
cholangiocellular
carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus
carcinoma,
cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical
carcinoma,
cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal
carcinoma,
encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides,
exophytic
carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniforni carcinoma,
gelatinous
carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular
carcinoma, granulosa cell
carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular
carcinoma, Hurthle cell
carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal
carcinoma,
carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma,
Krompecher's
carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular
carcinoma, carcinoma
lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma
medullare, medullary
carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma
muciparum,
carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous
carcinoma,
carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma
ossificans,
osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive
carcinoma, prickle
cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve
cell carcinoma,
carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma
scroti, signet-
ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid
carcinoma, spheroidal
cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous
carcinoma, squamous
cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma
telangiectodes,
transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma,
verrucous carcinoma, and
carcinoma villosum.
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103601 In certain embodiments the cancer is selected from lung
bronchioloalveolar carcinoma
(BAC), bladder cancer, a female genital tract malignancy (e.g., uterine serous
carcinoma,
endometrial carcinoma, vulvar squamous cell carcinoma, and uterine sarcoma),
an ovarian
surface epithelial carcinoma (e.g., clear cell carcinoma of the ovary,
epithelial ovarian cancer,
fallopian tube cancer, and primary peritoneal cancer), breast carcinoma, non-
small cell lung
cancer (NSCLC), a male genital tract malignancy (e.g., testicular cancer),
retroperitoneal or
peritoneal carcinoma, gastroesophageal adenocarcinoma, esophagogastric
junction carcinoma,
liver hepatocellular carcinoma, esophageal and esophagogastric junction
carcinoma, cervical
cancer, cholangiocarcinoma, pancreatic adenocarcinoma, extrahepatic bile duct
adenocarcinoma,
a small intestinal malignancy, gastric adenocarcinoma, cancer of unknown
primary (CUP),
colorectal adenocarcinoma, esophageal carcinoma, prostatic adenocarcinoma,
kidney cancer,
head and neck squamous carcinoma, thymic carcinoma, non-melanoma skin cancer,
thyroid
carcinoma (e.g., papillary carcinoma), a head and neck cancer, anal carcinoma,
non-epithelial
ovarian cancer (non-EOC), metastatic urothelial carcinoma (UC), uveal
melanoma, malignant
pleural mesothelioma, small cell lung cancer (SCLC), a central nervous system
cancer, a
neuroendocrine tumor, and a soft tissue tumor.
103611 In certain embodiments, the cancer is melanoma, non-small cell lung
cancer, colon
cancer, breast cancer, bladder cancer, or kidney cancer.
103621 In certain embodiments, the cancer is an adenocarcinoma. In certain
embodiments, the
cancer is a metastatic cancer. In certain embodiments, the cancer is a
refractory cancer.
103631 In certain embodiments, the cancer is resistant to or non-responsive to
treatment with
an antibody, e.g., an antibody with ADCC activity, e.g., avelumab.
103641 In certain embodiments, the cancer is a PD-L1-expressing cancer, e.g.,
the cancer
comprises cells that express PD-Li. An analysis of 196 tumor specimens from
patients with
renal cell carcinoma found that high tumor expression of PD-Li was associated
with increased
tumor aggressiveness and a 4.5-fold increased risk of death. High expression
of PD-Li is
associated with reduced numbers of tumor infiltrating lymphocytes and poor
prognosis. In
certain embodiments, the PD-Li status of a cancer can be determined using
immunohistochemistry staining protocols, such as DAKO 22C3 and VENTANA SP142
FDA
approved protocols, which are used as companion diagnostics for anti-PD-Li
antibodies
pembrolizumab, durvalumab, atezolizumab, and avelumab.
103651 The methods and compositions described herein can be used alone or in
combination
with other therapeutic agents and/or modalities. The term administered "in
combination," as
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used herein, is understood to mean that two (or more) different treatments are
delivered to the
subject during the course of the subject's affliction with the disorder, such
that the effects of the
treatments on the patient overlap at a point in time. In certain embodiments,
the delivery of one
treatment is still occurring when the delivery of the second begins, so that
there is overlap in
terms of administration. This is sometimes referred to herein as
"simultaneous" or "concurrent
delivery." In other embodiments, the delivery of one treatment ends before the
delivery of the
other treatment begins. In certain embodiments of either case, the treatment
is more effective
because of combined administration. For example, the second treatment is more
effective, e.g.,
an equivalent effect is seen with less of the second treatment, or the second
treatment reduces
symptoms to a greater extent, than would be seen if the second treatment were
administered in
the absence of the first treatment, or the analogous situation is seen with
the first treatment. In
certain embodiments, delivery is such that the reduction in a symptom, or
other parameter
related to the disorder is greater than what would be observed with one
treatment delivered in the
absence of the other. The effect of the two treatments can be partially
additive, wholly additive,
IS or greater than additive. The delivery can be such that an effect of the
first treatment delivered is
still detectable when the second is delivered.
103661 In certain embodiments, a method or composition described herein, is
administered in
combination with one or more additional therapies, e.g., surgery, radiation
therapy, or
administration of another therapeutic preparation. In certain embodiments, the
additional
therapy may include chemotherapy, e.g., a cytotoxic agent. In certain
embodiments the
additional therapy may include a targeted therapy, e.g., a tyrosine kinase
inhibitor, a proteasome
inhibitor, or a protease inhibitor. In certain embodiments, the additional
therapy may include an
anti-inflammatory, anti-angiogenic, anti-fibrotic, or anti-proliferative
compound, e.g, a steroid, a
biologic immunomodulator, a monoclonal antibody, an antibody fragment, an
aptamer, an
siRNA, an anti sense molecule, a fusion protein, a cytokine, a cytokine
receptor, a
bronchodilator, a statin, an anti-inflammatory agent (e.g., methotrexate), or
an NSAID. In
certain embodiments, the additional therapy may include a combination of
therapeutics of
different classes.
103671 In certain embodiments, a method or composition described herein is
administered in
combination with a second checkpoint inhibitor. The checkpoint inhibitor may,
for example, be
selected from a PD-1 antagonist, a second PD-Li antagonist, CTLA-4 antagonist,
adenosine
A2A receptor antagonist, B7-H3 antagonist, B7-H4 antagonist, BTLA antagonist,
KIR
antagonist, LAG3 antagonist, TIM-3 antagonist, VISTA antagonist or TIGIT
antagonist.
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103681 In certain embodiments, the checkpoint inhibitor is a PD-1 or a second
PD-Li
inhibitor. PD-1 is a receptor present on the surface of T-cells that serves as
an immune system
checkpoint that inhibits or otherwise modulates T-cell activity at the
appropriate time to prevent
an overactive immune response. Cancer cells, however, can take advantage of
this checkpoint
by expressing ligands, for example, PD-L1, that interact with PD-1 on the
surface of T-cells to
shut down or modulate T-cell activity. Exemplary PD-1/PD-L1 based immune
checkpoint
inhibitors include antibody based therapeutics. Exemplary treatment methods
that employ PD-
1/PD-L1 based immune checkpoint inhibition are described in U.S. Patent Nos.
8,728,474 and
9,073,994, and EP Patent No. 1537878B1, and, for example, include the use of
anti-PD-1
antibodies. Exemplary anti-PD-1 antibodies are described, for example, in U.S.
Patent Nos.
8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728,
9,102,727,
8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1
antibodies
include, for example, nivolumab (Opdivo , Bristol-Myers Squibb Co.),
pembrolizumab
(Keytruda , Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and
pidilizumab (CT-0I 1, Cure Tech). Exemplary anti-PD-L I antibodies are
described, for example,
in U.S. Patent Nos. 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and
8,217,149.
Exemplary anti-PD-Li antibodies include, atezolizumab (Tecentriq , Genentech),
durvalumab
(AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).
103691 In certain embodiments, a method or composition described herein is
administered in
combination with a CTLA-4 inhibitor. In the CTLA-4 pathway, the interaction of
CTLA-4 on a
T-cell with its ligands (e.g., CD80, also known as B7-1, and CD86) on the
surface of an antigen
presenting cells (rather than cancer cells) leads to T-cell inhibition.
Exemplary CTLA-4 based
immune checkpoint inhibition methods are described in U.S. Patent Nos.
5,811,097, 5,855,887,
6,051,227. Exemplary anti-CTLA-4 antibodies are described in U.S. Patent Nos.
6,984,720,
6,682,736, 7,311,910; 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797,
7,824,679,
8,143,379, 8,263,073, 8,318,916, 8,017,114, 8,784,815, and 8,883,984,
International (PCT)
Publication Nos. W098/42752, W000/37504, and W001/14424, and European Patent
No. EP
1212422 Bl. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.
103701 In certain embodiments, a method or composition described herein is
administered in
combination with a CTLA-4 inhibitor, e.g., a CTLA-4 inhibitor disclosed herein
103711 In certain embodiments, a method or composition described herein is
administered in
combination with an IDO inhibitor. Exemplary IDO inhibitors include 1-methyl-D-
tryptophan
(known as indoximod), epacadostat (INCB24360), navoximod (GDC-0919), and BMS-
986205.
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103721 Exemplary cytotoxic agents that can be administered in combination with
a method or
composition described herein include, for example, antimicrotubule agents,
topoisomerase
inhibitors, antimetabolites, protein synthesis and degradation inhibitors,
mitotic inhibitors,
alkylating agents, platinating agents, inhibitors of nucleic acid synthesis,
histone deacetylase
inhibitors (HDAC inhibitors, e.g., vorinostat (SAHA, MK0683), entinostat (MS-
275),
panobinostat (LBH589), trichostatin A (TSA), mocetinostat (MGCD0103),
belinostat (PXD101),
romidepsin (FK228, depsipeptide)), DNA methyltransferase inhibitors, nitrogen
mustards,
nitrosoureas, ethylenimines, alkyl sulfonates, triazenes, folate analogs,
nucleoside analogs,
ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epothilones,
intercalating agents,
agents capable of interfering with a signal transduction pathway, agents that
promote apoptosis
and radiation, or antibody molecule conjugates that bind surface proteins to
deliver a toxic agent.
In one embodiment, the cytotoxic agent that can be administered with a method
or composition
described herein is a platinum-based agent (such as cisplatin),
cyclophosphamide, dacarbazine,
methotrexate, fluorouracil, gemcitabine, capecitabine, hydroxyurea, topotecan,
irinotecan,
azacytidine, vorinostat, ixabepil one, bortezomib, taxanes (e.g., paclitaxel
or docetaxel),
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide,
vincristine, vinblastine, vinorelbine, colchicin, anthracyclines (e.g.,
doxorubicin or epirubicin)
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D,
adriamycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol,
puromycin, ricin, or maytansinoids.
103731 The invention also provides a method of increasing the expression of
HLA-DR, CD86,
CD83, IFN-y, IL-lb, IL-6, INFa, IL-17A, IL-2, or IL-6 in a cell, tissue, or
subject. The method
comprises contacting the cell, tissue, or subject with an effective amount of
an antibody, fusion
protein, and/or antibody conjugate, e.g., an antibody, fusion protein, or
antibody conjugate
disclosed herein. In certain embodiments, the cell is selected from a
dendritic cell and a
peripheral blood mononuclear cell (PBMC).
103741 In certain embodiments, expression of HLA-DR, CD86, CD83, IFN-y, IL-lb,
IL-6,
TNFa, IL-17A, IL-2, or IL-6 in the cell, tissue, or subject is increased by at
least about 10%, at
least about 20%, at least about 50%, at least about 75%, at least about 100%,
at least about
150%, at least about 200%, at least about 250%, at least about 300%, at least
about 400%, at
least about 500%, at least about 600%, at least about 700%, at least about
800%, at least about
900%, or at least about 1,000%, relative to a similar or otherwise identical
cell or tissue that has
not been contacted with the antibody, fusion protein, or antibody conjugate.
Gene expression
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may be measured by any suitable method known in the art, for example, by
ELISA, or by
Luminex multiplex assays.
103751 The invention also provides a method of promoting infiltration of
immune cells into a
tumor in a subject in need thereof. The method comprises administering to the
subject an
effective amount of an antibody, fusion protein, and/or antibody conjugate,
e.g., an antibody,
fusion protein, or antibody conjugate disclosed herein. In certain
embodiments, the immune
cells are T-cells, e.g., CD4+ and/or CD8+ T-cells, e.g., CD69 CD8+ and/or
Gzml3+CD8+ T-cells
In certain embodiments, the immune cells are natural killer (NK) cells.
103761 In certain embodiments, the infiltration of immune cells into the tumor
in the subject is
increased by at least about 10%, at least about 20%, at least about 50%, at
least about 75%, at
least about 100%, at least about 150%, at least about 200%, at least about
250%, at least about
300%, at least about 400%, at least about 500%, at least about 600%, at least
about 700%, at
least about 800%, at least about 900%, or at least about 1,000%, relative to a
similar or otherwise
identical tumor and/or subject that has not been administered the antibody,
fusion protein, or
antibody conjugate. Infiltration of immune cells into a tumor may be measured
by any suitable
method known in the art, for example, antibody staining.
103771 The invention also provides a method of increasing the number of
circulating natural
killer (NK) cells in a subject in need thereof The method comprises
administering to the subject
an effective amount of an antibody, fusion protein, and/or antibody conjugate,
e.g., an antibody,
fusion protein, or antibody conjugate disclosed herein, so as to increase the
number of
circulating NK cells relative to prior to administration of the antibody,
fusion protein, antibody
conjugate or pharmaceutical composition.
103781 In certain embodiments, the number of circulating NK cells in the
subject is increased
by at least about 10%, at least about 20%, at least about 50%, at least about
75%, at least about
100%, at least about 150%, at least about 200%, at least about 250%, at least
about 300%, at
least about 400%, at least about 500%, at least about 600%, at least about
700%, at least about
800%, at least about 900%, or at least about 1,000%, relative to a similar or
otherwise identical
subject that has not been administered the antibody, fusion protein, or
antibody conjugate.
Circulating NK cells in a subject may be measured by any suitable method known
in the art, for
example, antibody staining.
103791 The invention also provides a method of increasing the number of T-
cells in the
draining lymph node in a subject in need thereof The method comprises
administering to the
subject an effective amount of an antibody, fusion protein, and/or antibody
conjugate, e.g., an
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antibody, fusion protein, or antibody conjugate disclosed herein, so as to
increase the number of
T-cells in the draining lymph node relative to prior to administration of the
antibody, fusion
protein, antibody conjugate, or pharmaceutical composition. In certain
embodiments, the
immune cells are T-cells, e.g., CD4+ and/or CD8+ T-cells.
103801 In certain embodiments, the number of T-cells in the draining lymph
node in the
subject is increased by at least about 10%, at least about 20%, at least about
50%, at least about
75%, at least about 100%, at least about 150%, at least about 200%, at least
about 250%, at least
about 300%, at least about 400%, at least about 500%, at least about 600%, at
least about 700%,
at least about 800%, at least about 900%, or at least about 1,000%, relative
to a similar or
otherwise identical subject that has not been administered the antibody,
fusion protein, or
antibody conjugate, or pharmaceutical composition. T-cells in the draining
lymph node in a
subject may be measured by any suitable method known in the art, for example,
antibody
staining.
103811 The invention also provides a method of increasing expression of Cd3,
Cd4, Cd8,
Cd274, Ctla4, Icos, Pdcdl, Lag3, 116, Il lb, 112, Ifng, Ifnal, Mxl, Gzmb,
Cxcl9, Cxcl12, and/or
Cc15 in a cell, tissue, or subject. The method comprises contacting the cell,
tissue, or subject
with an effective amount of an antibody, fusion protein, and/or antibody
conjugate, e.g., an
antibody, fusion protein, or antibody conjugate disclosed herein, so as to
increase the expression
of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcdl, Lag3, 116, Il lb, 112, Ifng,
Ifnal, Mxl, Gzmb,
Cxcl9, Cxcl12, and/or Cc15 relative to the cell, tissue or subject prior to
contact with the
antibody, fusion protein, or antibody conjugate.
103821 In certain embodiments, expression of Cd3, Cd4, Cd8, Cd274, Ctla4,
Icos, Pdcdl,
Lag3, 116, Illb, 112, Ifng, Ifnal, Mxl, Gzmb, Cxcl9, Cxcl12, and/or Cc15 in
the cell, tissue, or
subject is increased by at least about 10%, at least about 20%, at least about
50%, at least about
75%, at least about 100%, at least about 150%, at least about 200%, at least
about 250%, at least
about 300%, at least about 400%, at least about 500%, at least about 600%, at
least about 700%,
at least about 800%, at least about 900%, or at least about 1,000%, relative
to a similar or
otherwise identical cell, tissue, or subject that has not been contacted with
the antibody, fusion
protein, or antibody conjugate. Gene expression may be measured by any
suitable method
known in the art, for example, by ELISA, Luminex multiplex assays, or
Nanostring technology.
103831 The invention also provides a method of removing sialic acid from a
cell or tissue. The
method comprises contacting the cell or tissue with an effective amount of a
fusion protein
and/or antibody conjugate, e.g., a fusion protein or antibody conjugate
disclosed herein. The
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invention also provides a method of removing sialic acid from a cell in a
subject, the method
comprising administering to the subject an effective amount of a
pharmaceutical composition
comprising a fusion protein and/or antibody conjugate, e.g., a fusion protein
or antibody
conjugate disclosed herein, thereby to remove sialic acid from the cell.
103841 In certain embodiments, the cell is tumor cell, dendritic cell (DC)
or monocyte. In
certain embodiments, the cell is a monocyte, and the method results in
increased expression of
an 1VIFIC-II molecule (e.g., BLA-DR) on the monocyte. In certain embodiments,
expression of
an MHC-II molecule in the cell or tissue is increased by at least about 10%,
at least about 20%,
at least about 50%, at least about 75%, at least about 100%, at least about
150%, at least about
200%, at least about 250%, at least about 300%, at least about 400%, at least
about 500%, at
least about 600%, at least about 700%, at least about 800%, at least about
900%, or at least about
1,000%, relative to a similar or otherwise identical cell or tissue that has
not been contacted with
the fusion protein and/or antibody conjugate. Gene expression may be measured
by any suitable
method known in the art, for example, by ELISA, by Luminex multiplex assays,
or by flow
cytometry.
103851 The invention also provides a method of enhancing phagocytosis of a
tumor cell. The
method comprises contacting the tumor cell with a fusion protein and/or
antibody conjugate,
e.g., a fusion protein or antibody conjugate disclosed herein, in an amount
effective to remove
sialic acid from the tumor cell, thereby enhancing phagocytosis of the tumor
cell. In certain
embodiments, the disclosure relates to a method of increasing phagocytosis of
a tumor cell in a
subject, the method comprising administering to the subject an effective
amount of a
pharmaceutical composition a fusion protein and/or antibody conjugate, e.g., a
fusion protein or
antibody conjugate disclosed herein, in an amount effective to remove sialic
acid from the tumor
cell, thereby to increase phagocytosis of the tumor cell.
103861 In certain embodiments, phagocytosis is increased by at least about
10%, at least about
20%, at least about 50%, at least about 75%, at least about 100%, at least
about 150%, at least
about 200%, at least about 250%, at least about 300%, at least about 400%, at
least about 500%,
at least about 600%, at least about 700%, at least about 800%, at least about
900%, or at least
about 1,000%, relative to a similar or otherwise identical tumor cell or
population of tumor cells
that has not or have not been contacted with the fusion protein and/or
antibody conjugate.
Phagocytosis may be measured by any suitable method known in the art.
103871 The invention also provides a method of activating a dendritic cell
(DC). The method
comprises contacting the DC with a tumor cell that has been treated with a
fusion protein and/or
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antibody conjugate, e.g., a fusion protein or antibody conjugate disclosed
herein. In certain
embodiments, the disclosure relates to a method of activating a dendritic cell
(DC) or a
population of DCs in a subject, the method comprising administering to the
subject an amount of
a pharmaceutical composition comprising a fusion protein and/or antibody
conjugate, e.g., a
fusion protein or antibody conjugate disclosed herein, effective to remove
sialic acid from a
tumor cell in the subject, thereby to activate the DC or the population of DCs
in the subject.
103881 In certain embodiments, activation of the DC or a population of DCs is
increased by at
least about 10%, at least about 20%, at least about 50%, at least about 75%,
at least about 100%,
at least about 150%, at least about 200%, at least about 250%, at least about
300%, at least about
400%, at least about 500%, at least about 600%, at least about 700%, at least
about 800%, at
least about 900%, or at least about 1,000%, relative to a similar or otherwise
identical DC or
population of DCs that has not or have not been contacted with a tumor cell
that has been treated
with the fusion protein and/or antibody conjugate. Activation may be measured
by any suitable
method known in the art.
103891 The invention also provides a method of reducing Siglec-15 binding
activity, thereby
to increase anti-tumor activity in a tumor microenvironment, the method
comprising contacting
a T cell with an antibody, fusion protein, and/or antibody conjugate, e.g., an
antibody, fusion
protein, or antibody conjugate disclosed herein. In certain embodiments, the
disclosure relates
to a method of reducing Siglec-15 binding activity, thereby to increase anti-
tumor activity in a
tumor microenvironment of a patient, the method comprising administering to
the subject an
effective amount of a pharmaceutical composition comprising an antibody,
fusion protein,
and/or antibody conjugate, e.g., an antibody, fusion protein, or antibody
conjugate disclosed
herein, thereby to increase anti-tumor activity (e.g., T cell activity) in the
subject.
103901 In certain embodiments, Siglec-15 binding activity is reduced by at
least about 10%, at
least about 20%, at least about 50%, at least about 75%, or about 100%,
relative to Siglec-15
that has not or have not been contacted with the antibody, fusion protein,
antibody conjugate,
and/or pharmaceutical composition. Binding may be measured by any suitable
method known
in the art.
103911 Throughout the description, where compositions are described as having,
including, or
comprising specific components, or where processes and methods are described
as having,
including, or comprising specific steps, it is contemplated that,
additionally, there are
compositions of the present invention that consist essentially of, or consist
of, the recited
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components, and that there are processes and methods according to the present
invention that
consist essentially of, or consist of, the recited processing steps.
103921 In the application, where an element or component is said to be
included in and/or
selected from a list of recited elements or components, it should be
understood that the element
or component can be any one of the recited elements or components, or the
element or
component can be selected from a group consisting of two or more of the
recited elements or
components
103931 Further, it should be understood that elements and/or features of a
composition or a
method described herein can be combined in a variety of ways without departing
from the spirit
and scope of the present invention, whether explicit or implicit herein. For
example, where
reference is made to a particular compound, that compound can be used in
various embodiments
of compositions of the present invention and/or in methods of the present
invention, unless
otherwise understood from the context. In other words, within this
application, embodiments
have been described and depicted in a way that enables a clear and concise
application to be
written and drawn, but it is intended and will be appreciated that embodiments
may be variously
combined or separated without parting from the present teachings and
invention(s). For
example, it will be appreciated that all features described and depicted
herein can be applicable
to all aspects of the invention(s) described and depicted herein.
103941 It should be understood that the expression "at least one of' includes
individually each
of the recited objects after the expression and the various combinations of
two or more of the
recited objects unless otherwise understood from the context and use. The
expression -and/or"
in connection with three or more recited objects should be understood to have
the same meaning
unless otherwise understood from the context.
103951 The use of the term "include," "includes," "including," "have," "has,"
"having,"
"contain," "contains," or "containing," including grammatical equivalents
thereof, should be
understood generally as open-ended and non-limiting, for example, not
excluding additional
unrecited elements or steps, unless otherwise specifically stated or
understood from the context.
103961 Where the use of the term "about" is before a quantitative value, the
present invention
also includes the specific quantitative value itself, unless specifically
stated otherwise. As used
herein, the term "about- refers to a 10% variation from the nominal value
unless otherwise
indicated or inferred.
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103971 It should be understood that the order of steps or order for performing
certain actions is
immaterial so long as the present invention remain operable. Moreover, two or
more steps or
actions may be conducted simultaneously.
103981 The use of any and all examples, or exemplary language herein, for
example, "such as"
or "including," is intended merely to illustrate better the present invention
and does not pose a
limitation on the scope of the invention unless claimed. No language in the
specification should
be construed as indicating any non-claimed element as essential to the
practice of the present
invention.
EXAMPLES
103991 The following Examples are merely illustrative and are not intended to
limit the scope
or content of the invention in any way.
Example 1
104001 This example describes the construction of recombinant human sialidases
(Neul, Neu2,
and Neu3)
104011 The human sialidases Neul, Neu2, Neu3 (isoform 1), and Neu4 (isoform 1)
were
expressed as secreted proteins with a 10xHis tag. To express Neul as a
secreted protein, the
native N terminal signal peptide
(MTGERPSTALPDRRWGPRILGFWGGCRVWVFAAIFLLLSLAASWSKA, SEQ ID NO: 27)
was replaced by MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28), and the C terminal
lysosomal signal motif (YGTL; SEQ ID NO: 29) was removed. To express Neu2,
Neu3, and
Neu4 as secreted proteins, the N terminal signal peptide
MDMRVPAQLLGLLLLWLPGARC
(SEQ ID NO: 28) was added to each.
104021 Sialidases were expressed in a 200 mL transfection of HEK293F human
cells in 24-
well plates using the pCEP4 mammalian expression vector with an N-terminal
6xHis tag.
Sialidases were purified using Ni-NTA columns, quantified with a UV-Vis
spectrophotometer
(NanoDrop), and examined by SDS-PAGE as shown in FIGURE 1. Neul expressed
well, with
a yield of ¨3 [ig/ml, and was present primarily in a monomeric form. Neu2 and
Neu3 expression
each gave yields of ¨0.15 tig/mL and each were present primarily in a dimeric
form. Neu4 had
no detectable expression yield as measured by NanoDrop. Bacterial sialidase
from Salmonella
typhimurium (St-sialidase, SEQ ID NO: 30), which was used as a positive
control for expression,
gave a comparable yield to Neul, and was present primarily in a monomeric
form.
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104031 The activity of the recombinantly expressed sialidases was assayed by
measuring the
release of sialic acid from the fluorogenic substrate 4-methylumbelliferyl-N-
acetylneuraminic
acid (4MU-NeuAc). As shown in FIGURE 2, Neul has no detectable activity above
a no-
enzyme control, which is consistent with previous reports indicating that Neul
is inactive unless
it is in complex with beta-galactosidase and protective protein/cathepsin A
(PPCA). Neu2 and
Neu3 were active. An enzyme kinetics assay was performed with Neu2 and Neu3. A
fixed
concentration of enzyme at 1 nM was incubated with fluorogenic substrate 4MU-
NeuAc at
concentrations ranging from 4000 uM to 7.8 tM. Assays were conducted at both
acidic (pH
5.6) and neutral (pH 7) conditions. As shown in FIGURE 3, both Neu2 and Neu3
were active at
acidic and neutral conditions and showed enzyme kinetics that were comparable
to those
previously reported.
104041 Most of the recombinantly expressed sialidases ran as aggregates or
dimers on a non-
reducing SDS-PAGE gel. Subsequent treatment with the reducing agent
dithiothreitol (DTT)
resulted in a monomeric form of the enzyme that ran at 42 kDa on a reducing
SDS-PAGE gel
(FIGURE 1).
Example 2
104051 This example describes PD-Li antibody discovery and hybridoma
screening.
104061 Antibodies were generated using two different methods. In the first
method (Green
Mountain Antibody, Vermont), 3 SJL/J mice and BALB/cJ mice were immunized
using hPD-
Ll-hFc following the 28-day REVIMS protocol. PEG fusion of splenocytes and
lymphocytes
from the high titer mice with NS1 myeloma cells was performed to generate
hybridomas.
104071 In the second method (Aldevron, WI), human PD-Li extracellular domain
(ECD) was
cloned into a vector plasmid also containing a detection tag for immunization
and control. The
plasmid constructs were transfected into mammalian cells. hPD-L1 expression
was validated
using flow cytometry with an hPD-L1 antibody and anti-tag antibody, the vector
control using an
irrelevant anti-tag antibody. Five mice were immunized with validated PD-Li
ECD plasmid
DNA. The immune response was checked with mice sera using flow cytometry on
cells
transfected with hPD-Ll. PEG fusion of splenocytes and lymphocytes from the
high titer mice
with NS1 myeloma cells was performed to generate hybridomas.
104081 Hybridoma supernatants were screened using an ELISA to determine
binding to human
PD-L1 (hPD-L1) as well as cynomolgus PD-L1 (cPD-L1). Hybridoma supernatants
were
diluted 10x in ELISA binding buffer prior to loading on a hPD-L1-his tagged or
cPD-L1-his
tagged coated ELISA wells. The binding of mouse IgGs were detected using HRP-
conjugated
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Goat-anti-mouse polyclonal antibody. The plate was developed with TMB and Stop
buffer, the
absorbance at 450 nm was read using SpectraMax plate reader.
104091 A second assay was also utilized wherein hybridoma supernatants were
screened for
the ability to block biotinylated hPD-1-Fc from binding to hPD-L1. Hybridoma
supernatants
were diluted 3x in ELISA binding buffer and mixed with biotin-hPD-1-Fc at a
final
concentration of 1 u.g/mL. The mixtures were loaded to hPD-L1-Fc coated ELISA
wells for
binding. The antibodies that recognized the hPD-1/hPD-L1 epitope bin competed
for binding
and reduced the hPD-1-Fc binding signal. The residual binding of biotin-hPD-1-
Fc to hPD-Ll-
Fc was detected with EIRP conjugated Streptavidin. The plate was developed
with TMB and
Stop buffer, and the absorbance at 450 nm was read using SpectraMax plate
reader. The A450
absorbance was normalized to the hybridoma-conditioned medium control.
104101 TABLE 10 is a summary of representative hybridoma supernatant screening
results.
Selected clones with good binding to hPD-L1 and cPD-L1 and with low residual
binding were
further characterized.
TABLE 10 Summary of Hybridoma Supernatant Screening
Clone huPD-L1-His cyPD-L1-His Block Residual %
01A11 0.04325 0.0444 68%
01E09 2.36185 1.5722 35%
01G01 0.0496 0.0484 85%
01G07 1.9901 1.3883 11%
02C01 1.8316 1.3126 11%
02CO3 0.05075 0.04525 94%
02C04 2.06255 1.441 47%
02D06 0.03525 0.0367 87%
02D10 1.83325 1.36395 9%
02E09 1.96075 1.27385 82%
02F06 0.0491 0.0451 92%
02G09 0.04745 0.0418 89%
02H01 0.1207 0.08695 91%
02H02 0.0385 0.0375 99%
02H07 2.3958 1.46115 30%
03D03 0.0425 0.0402 84%
03G01 0.04025 0.04055 82%
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Clone huPD-LI-His cyPD-LI-His Block Residual A
03G08 0.04285 0.04215 93%
03H06 0.04915 0.04295 92%
04C01 0.0441 0.041 92%
04C11 1.996 1.07865 33%
04D05 2.1616 1.3421 39%
04G02 0.0483 0.0447 89%
04H04 0.04255 0.0387 82%
05A04 0.03905 0.03935 82%
05CO2 0.03905 0.0385 88%
05D01 0.04775 0.0431 79%
05F02 0.0395 0.0398 81%
05G01 0.06305 0.04815 96%
05H01 0.04225 0.0419 83%
051103 0.0427 0.04095 83%
05H07 0.05695 0.0472 81%
06A10 0.0427 0.04205 102%
06H03 0.0386 0.0392 99%
07C04 2.5075 1.385 43%
07D11 0.037 0.03705 99%
07G03 0.23005 0.17695 112%
07G09 2.57165 1.49485 119%
08F01 0.03855 0.0361 100%
08F02 0.97025 1.13735 106%
08F08 0.0378 0.03775 101%
09B07 1.706 0.5715 72%
09C01 2.61775 1.31865 18%
09F04 0.04145 0.04025 103%
09F05 0.0412 0.04235 116%
09F07 2.5844 1.31555 46%
09G07 0.04155 0.03915 123%
10B03 0.0439 0.0402 109%
10B04 0.0445 0.04275 114%
10007 0.04485 0.04475 118%
10008 3.2374 0.12175 17%
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Clone huPD-LI-His cyPD-LI-His Block Residual %
10D07 0.0387 0.03835 118%
10E05 0.04135 0.04345 112%
10E06 2.2714 1.5315 106%
10F03 0.03975 0.04215 118%
10F04 0.04305 0.03955 118%
10F05 0.04405 0.04045 97%
10F07 1.581 1.36875 101%
10F10 0.0412 0.0387 104%
10G09 2.75445 1.2858 51%
11A01 0.0504 0.0437 111%
11A06 0.04655 0.0439 111%
11A08 2.049 1.4734 107%
11F01 2.2419 1.62365 116%
111103 2.9876 1.32625 70%
12A02 0.35785 0.4006 76%
12A05 2.42665 1.40435 47%
12B11 2.07995 1.2203 51%
12E08 2.0973 1.46875 32%
12G04 2.1039 1.37465 26%
121104 2.05395 1 4835 75%
121-106 2.0525 1.42365 109%
13F05 2.3518 1.4181 6%
13G07 0.3502 0.6019 69%
13H02 2.2304 1.35665 22%
14B06 0.0383 0.0414 86%
14C09 1.2704 1.12745 80%
14F11 0.0405 0.04175 80%
14G07 2.2582 1.4165 40%
141107 1.57355 1.31875 64%
15D01 2.41115 1.4562 37%
15D05 0.04585 0.0435 86%
15E01 0.04575 0.04655 82%
15F06 0.9242 0.78545 111%
15G01 0.0506 0.04365 87%
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Clone huPD-LI-His cyPD-LI-His Block Residual A
15G09 0.0423 0.04545 83%
15H04 2.221 1.4642 21%
15H08 0.05165 0.0468 74%
16A04 0.0475 0.0514 67%
16B05 0.183 0.16385 76%
16F01 0.64805 0.94295 65%
16F08 2.4565 1.32575 39%
16G01 2.3645 0.9678 7%
16H04 0.0439 0.04285 82%
POS 2.31 1.44 10%
NEG 0.034 0.03475 60%
104111 Functional blocking assay. The supernatants from selected PD-Li
antibody-
producing hybridomas were tested in co-culture with (i) engineered CHO-Kl
cells expressing
human PD-Li and TCR activating protein and (ii) Jurkat T cells expressing
human PD-1, TCR
and a luciferase reporter driven by an NEAT response element. Absent
intervention, PD-Li
interacting with PD-1 inhibits TCR-mediated luminescence. Blockade of the PD-
Li! PD-1
interaction, for instance using avelumab or PD-Li antibody-containing
supernatants, results in a
luminescent signal. A luciferase substrate was added after 6 hours of
incubation and
luminescence was measured. Relative light units (RLU) were calculated by
subtracting
background (substrate and media only) from assay wells. Fold induction was
calculated by
dividing the RLU of induced cells minus background by the RLU of the no
antibody control
minus background. As shown in FIGURE 7, hybridoma supernatants capable of
disrupting the
PD-Ll/PD-1 interaction, resulting in a dose dependent increase in
luminescence, were identified.
FIGURES 7A, 7B and 7C represent the testing of three batches antibodies.
Avelumab was used
as a positive control producing a Fold Induction of 4 to 5 over a range of 0.1
to 10 ug/mL in this
assay (data not shown). Fold induction = RLU (induced - background) / RLU (no
antibody
control - background). EC5os of the indicated PD-Li antibodies were comparable
to the EC50 for
avelumab.
104121 Screening of purified hybridoma antibodies. ForteBio octet binding of
purified
antibodies to recombinant hPD-Ll-his and cPD-Ll-his was measured. Mouse IgGs
purified
from hybridoma supernatant were captured on AMC (Anti-mouse IgG-Capture)
Biosensor.
hPD-L 1 -hi s or cPD-Ll-his analytes were titrated from 100 nM in a 2x series
dilution. The signal
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was subtracted with buffer reference and aligned to the baseline. KD, Kon and
Koff values were
generated using a 1:1 fitting model. The binding kinetics of selected purified
hybridomas are
shown in FIGURES 8A, 8B, 8C, and 80 for hPD-L1 and FIGURES 9A, 9B, 9C, and 90
for
cPD-L1. The X-axes represent assay time in seconds, and the Y-axes represent
binding signal
on the biosensor. Each line represents the real time signal of antigen
association and
dissociation at the given antigen concentration in the assay (e.g., the top
line represents the
signal of the highest antigen concentration in the assay, the second top line
represents the second
highest concentration in the assay). The vertical dashed line represents the
time point that the
assay was moved from association step to the dissociation step. All the
ForteBio/Octet assays
were using standard/conventional settings and the graphs provided in the
figures will be
understood by one of skill in the art. The calculated KDs are shown in TABLE
11 for hPD-L1
and cPD-LI.
104131 Purified hybridoma antibodies were tested for their ability to block
biotinylated hPD-1-
Fc from binding to hPD-L1. Antibodies were 3x titrated and mixed with
biotinylated hPD-1-Fc
at final concentration of 1 ug/mL. The mixture of antibody and biotin-hPD-1-Fc
were loaded to
hPD-L1-Fc coated ELISA wells for binding. The antibodies that recognize the
hPD-1/hPD-L1
epitope bin will compete for the binding and result hPD-1-Fc binding signal
reduction. The
residual binding of biotin-hPD-1-Fc to hPD-L1-Fc were detected with HRP
conjugated
Streptavidin. The plate was developed with TMB and Stop buffer, the absorbance
at 450 nm
was read using SpectraMax plate reader. The A450 absorbance was normalized to
the control
that without antibody for the percentage values. The curve and ICsowere
generated using
GraphPad Prism software. As depicted in FIGURE 10, antibodies demonstrated
either complete
(C) or partial (P) blocking of hPD-1 to hPD-L1. The IC50 as determined from
the data is shown
in FIGURE 10 with many in the low single nM range or lower and is also shown
in TABLE 11.
TABLE 11 Summary of anti-hPD-L1 hybridoma antibodies
cell Kinetics KD (M) ELISA
block
Hybridoma PAL ID C/P hPD-L1 cPD-L1 cyno, Fold IC50,
nM C/P
over Bkg
1D9.A7 PAL747 C Low signal Low signal 1.10 1.69*
1D9.B6 PAL748 Low signal 0.94
3E2.B6 PAL749 P 9.92x10' 7.44x10' 43.49 1.65
4G6.A3 PAL751 C 3.85x10-th
4G6.H4 PAL 752 7.89x10-1 4.03x10' 18.39
2.53
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cell Kinetics KD (M) ELISA
block
13F1.B7 PAL759 C 1.14x10 9 13.12 4.64*
13F1.F2 PAL760 7.11x104 1.09x10 9 32.82
1G7.A8 PAL767 C
8.03x101 5.77x1011 36.91 0.81
1G7.C8 PAL768 5.59x101 34.91
2C1.C8 PAL769 C 7.58x104 1.3x109 39.52 0.51
2C1.D11 PAL770 8.82x101 36.07
2D10E2.B9 PAL771 C 5.92x10A 1.15x10'
2D10.E2.H4 PAL771.2 1.54x10- 9 2.27x10- 9 38.14 0.6
2D10.G3 PAL772 1.75x10- 9 2.64x1e9 22.16
1008.A7 PAL775 P 2 phase 1.82 2.37
1008.H5 PAL776 2 phase 1.15
16G1.A5 PAL785 C 1.02x10- 9 6.42x10-1 4.98 0.68
16G1.D7 PAL786 1.45x10- 9 1.73x10- 9 2.97
CKH-3D10 PAL787(3D10) P 2.77x10-th 2.83x10A 38.22 4.07
CKH-3E3 PAL788(3E3) C 1.88x101 3.92x104 24.81 0.89
Avclumab C 7.54x101 1.7x10' N/A 0.38
Example 3
104141 This example describes the generation and characterization of chimeric
PD-Li
antibodies. VH and VL gene sequences of hybridoma antibodies were isolated and
sequenced.
VL sequences are shown in TABLE 12, VH sequences in TABLE 13 and Light and
Heavy
Chain CDRs in TABLE 14. The DNA fragments coding for the V gene of interest
were
synthesized through conventional vendor. The VH and VL sequences were cloned
into human
IgG1 constant heavy chain backbone and human constant kappa light chain
backbone
respectively. The heavy chain and light chain DNA plasmids were transiently co-
transfected in
HEK293 cells to express the full IgGs.
TABLE 12. Encoded Variable Light sequences
ID VL
PAL DIQMTQSSFSFSVSLGDRVTIICKASEDIYNRLAWYQQKPGNTPRLLISGATSLETGVPSRFSGSGS
752 GKDYTLSITSLQTEDVATYYCQQYWSTPWTEGGGTKLEIK (SEQ ID NO: 136)
PAL DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGT
759 DFTLSINSVESEDIGDYYCQQSNNWPFTEGSGTKLEIK (SEQ ID NO: 144)
PAI, DTVMTQSP A ST ,AVST ,GOK A TTSCK A SKKVTTECISTSVLHWYOOKPGQPPKI ,TYNCIAKT
,FSGVS A R
760 FSDSGSQNRSPEGNQLNFTLTIDPVEADDAATYYCLQNKEVPYTEGGGTELEIK (SEQ ID
NO:
152)
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PAL DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNSLAWYQQKPGQSPKLLIYWASTRESGV
767 PDRETGSGSGTDFTLTISSVKAEDLAVYYCQQYYGYPWTEGGGTKLEIK (SEQ ID NO:
160)
PAL SIVMTQTPKELLVSAGDRVTITCKASQSVSNDVIWYQQKPGQSPKLLIYYASIRFTGVPDREAGS
769 GYGTDFTFTINTVQAEDLAVYFCQQDYNSPWTEGGGTKLEIK (SEQ ID NO: 167)
PAL QIVLTQ SPAIMSASP GEKVTMTC SAS S SVSYMYWYQQKPGSSPRLLIYDTSNLAS
GVPLRFS GS G
771 SGTSYSLTLSRMEAEDAATYYCQQWSTYPLTFGAGTKLELK (SEQ ID NO: 174)
PAL DIVLTQSPASLAVSLGQRATISCRASESVEFYGTTLMQWYQQKPGQPPKLLIYAASNVES
GVPAR
785 FSGSGSGTDFSLNIHPVEEGDIGMYFCQQSRKVPYTEGGGTKLEIK (SEQ ID NO: 182)
PAL DIVMTQSQNEMSTSVGDRVSVTCKASHYVGTEVAWYQQKPGQSPKALIFSTSYRHTGVPDRFT
787 GSGSGTDFTLTISNVQSEDLADYFCQQYYNSPLTFGAGTKLELK (SEQ ID NO: 190)
PAL NIVLTQ SPA SLAV SL GQRATI S CRA SE S VD SY GN SFMHWYQ QKP
GQPPKLLTYLA S NLQ S GVPAR
788 FSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPWTEGGGTKLEIK (SEQ ID NO: 198)
TABLE 13. Encoded Variable Heavy sequences
ID VH
PAL EVQL QE S GAEL ARP GA S VKL S CKA S GHAFT SD S INWVKQRI GQ GLE WI GEIYPRS
GNPYYNEKFK
752 GKATLTADKS S S TAYMELR SLT S ED SAVYFCATDYYGRYFDVWGTGTTVTVS S (
SEQ ID NO:
132)
PAL EVQLQESGAELVRPGASVKL SCKA SGY SFTDYYINWVKQRPGQGLEWIARTYP GSGNTYYNEKF
759 KGKATLTAEKSSITAYMQL SSLTSED S AVYF CAR SYYY G S SYLFDYW GQ GTTL
TV S S (SEQ ID
NO: 140)
PAL EVQLQQSGPELVKPGALVKISCKASGYTFTDYYMNWVKKSHGRSLEWIGDINPNNGYTNYNQN
760 FKGKATLTVDKS S STVYMELRSLTSED S AVYY C AR S AAYYVLDD WGQ GT SVTVS
S ( SEQ ID NO:
148)
PAL EVQL QE S GP SL VKP SQTL SLTCSVTGD SIT S GYWNWT RKFP GNKLEYMGYI SYTG
STYYNP SLKR
767 RISITRDTSKNQYYLQLNSVTTEDTATYYCASQGGWLQAMDYWGQGTSVTVSS (SEQ ID NO:
156)
PAL EVQLQESGAELVKPGASVTLSCTASGENIKDTYMHWVKQRPEQGLEWIGRIDPANDNTKYDPKE
769 QDKATITADTS SD TAYLRL S S LT SED TAVYYCAREGY GG SY GEGYW GQ
GTTLTV S S (SEQ ID
NO: 164)
PAL EVQLQESGAELVKPGASVKLSCTASGENIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKE
771 PGKATITADTS SNTAYLQL S S LT SED AAVYY CARPFNYRFYD VYYFDYWGQ
GTTLTV S T (SEQ
ID NO: 170)
PAL EVQLQESGPELVKPGTSVKMSCKASGYTFTSYVMHWVKQRPGQGLEWIGYINPYNDGSKYNEK
785 FKGKATLTSDTSSSTAYMELSSLTSEDSAVYYCAKQTLDFWGQGTSVTVST (SEQ ID NO:
178)
PAL QVTLKESGPGILQPSQTLSLTCSFSGESLSTYGLGVGWIRQPSGKGLEWLANIWWNDDKEYDSVL
787 KSRLTISKDTSNNQVFLKISSVDTSETATYYCAQTLHYYDGIAWFAYWGQGTLVTVSA (SEQ
ID
NO: 186)
PAL QVQLQQPGAELVKP GA SVKL S CKASGYTFTSNWMNWVKQRPGRGLEWIGRIHP SD SETHYHQK
788 FKSKATLTVDKS S STAYIQLS SLTSED SAVYYCAHSSGDYGRDYWGQGTTLTVS S (
SEQ ID NO:
194)
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TABLE 14. Light and Heavy Chain CDRs
ID CDR-L1 CDR-L2 CDR-L3 CDR-H1 CDR-H2 CDR-H3
PAL752 EDIYNR GAT (SEQ QQYWSTPWT GHAFTSDS 1YPRSGNP
DYYGRYFD
(SEQ ID NO: ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID
NO: V (SEQ ID
133) 134) 135) 129) 130)
NO: 131)
PAL759 QSIGTS (SEQ YAS (SEQ QQSNNWPFT GYSFTDYY IYPGSGNT
SYYYGSSYL
ID NO: 141) ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID
NO: FDY (SEQ ID
142) 143) 137) 138)
NO: 139)
PAL760 KKVTIFGSIS NGA (SEQ LQNKEVPYT GYTFTDYY INPNNGYT
SAAYYVLD
V (SEQ ID ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID
NO: D (SEQ ID
NO: 149) 150) 151) 145) 146)
NO: 147)
PAL767 QSLLYSSNQ WAS (SEQ QQYYGYPWT GDSITSGY ISYTGST
QGGWLQAM
KNS (SEQ ID ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID
NO: DY (SEQ ID
NO: 157) 158) 159) 153) 154)
NO: 155)
PAL769 QSVSND YAS (SEQ QQDYNSPWT GFNIKDTY IDPANDNT
EGYGGSYGE
(SEQ ID ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID
NO: GY (SEQ ID
NO:165) 142) 166) 161) 162)
NO: 163)
PAL771 SSVSY (SEQ DTS (SEQ QQWSTYPLT GFNIKDTY IDPANGNT
PFNYRFYDV
ID NO: 171) ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID
NO: YYFDY (SEQ
172) 173) 161) 168)
ID NO: 169)
PAL785 ESVEFYGTT AAS (SEQ QQSRKVPYT GYTFTSYV INPYNDGS
QTLDF (SEQ
L (SEQ ID ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID
NO: ID NO: 177)
NO: 179) 180) 181) 175) 176)
PAL787 HYVGTF STS (SEQ QQYYNSPLT GFSLSTYGLG IWWNDDK TLHYYDGIA
(SEQ ID NO: ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID
NO: WFAY (SEQ
187) 188) 189) 183) 184)
ID NO: 185)
PAL788 ESVDSYGNS LAS (SEQ QQNNEDPWT GYTFTSNW IHPSDSET
SSGDYGRDY
F (SEQ ID ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID
NO: (SEQ ID NO:
NO: 195) 196) 197) 191) 192)
193)
104151 Characterization of chimeric anti-PD-Li antibodies. ForteBio octet
binding of
purified chimeric antibodies and recombinant hPD-Ll-his and cPD-Ll-his was
measured.
Chimeric human antibodies were captured on AHC (Anti-human IgG-Capture)
Biosensor, hPD-
LI-his or cPD-Li-his analytes were titrated from 100 nM in a 2x series
dilution. The buffer
reference was subtracted from the signal and aligned to the baseline. KD, Kon
and Koff values
were generated using 1:1 fitting model as described above. The binding
kinetics of selected
chimeric antibodies are shown in FIGURE 11A and FIGURE 11B for hPD-L1 and
FIGURE
11C and FIGURE 11D for cPD-L1. The calculated KDs are shown in TABLE 15 for
hPD-L1
and cPD-L1.
104161 Purified chimeric antibodies were tested for their ability to block
biotinylated hPD-1-Fc
from binding to hPD-L1 as described above. FIGURE 12 depicts the results and
the calculated
ICsos, all of which are in the single digit nM range. Protein A purified
chimeric antibodies were
examined by size exclusion chromatography and quantified with a UV-Vis
spectrophotometer
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(NanoDrop; mAU=milli absorbance units). FIGURE 13 depicts UV traces of size
exclusion
chromatographs with the indicated amount of monomeric peaks at the expected
retention times.
[0417] Purified chimeric antibodies were evaluated for their ability to bind
PD-Li expressed
on two human cancer cell lines. HCC827 and NCI-292 cells were incubated with
titrated
antibodies for 30 min at 4 C. Cells were washed and incubated with AF647-
labeled goat anti-
human IgG(H+L) for 30 min at 4 C. Cells were washed, fixed and analyzed on
FACSCelesta.
FIGURE 14 depicts the binding curves and calculated Kd for selected chimeric
antibodies to
HCC827 cells (FIGURE 14A) and NCI-292 cells (FIGURE 14B).
[0418] Purified chimeric antibodies were evaluated for their ability to bind
and be internalized
by human dendritic cells (DC). Monocyte-derived DC (moDC) were either (1)
stimulated with
Pam3CSK4 at 250 ng/ml before the day of experiment or (2) not stimulated.
Cells were blocked
for 30 min at room temperature, and incubated with 1 nM or 10 nM antibodies
for 30 min on ice.
Cells were washed and divided into two equal portions for a 2-hour incubation
on either ice or at
37 C. Cells were washed and incubated with goat anti-human IgG(H+L) for 30
min on ice.
Cells were washed, fixed, and analyzed on FACSCelesta. Percent internalization
was
determined as the reduction of bound antibody on cells after 2 hour incubation
at 37 'V as
compared to 4 C. FIGURE 15 depicts the % internalization for the indicated
chimeric
antibodies under the different conditions. All antibodies had a relatively
limited rate of
internalization, between 20% and 30%, after 2 hours including with the
stimulated cells.
[0419] Purified chimeric antibodies were evaluated for their ability to
functionally block PD-1
binding to PD-Li as described above. Purified chimeric PD-Li antibodies were
tested in co-
culture with (i) engineered CHO-Kl cells expressing human PD-Ll and TCR
activating protein
and (ii) Jurkat T cells expressing human PD-1, TCR and a luciferase reporter
driven by an NFAT
response element. As shown in FIGURE 16, the tested chimeric antibodies
disrupted the PD-
Li/PD-1 interaction, resulting in a dose dependent increase in luminescence.
Kds of the PD-Li
antibodies were all single digit nM or sub nM.
[0420] The specificity of the antibodies for cell surface expressed hPD-L1 was
confirmed by
comparing binding of antibodies to CHO cells expressing hPD-L1 vs wild type
CHO cells.
CHO cells were incubated with 100 nM antibodies and CHO-PD-Li cells (from
Bioassay) with
10 nM antibodies for 30 min at 4 C. Cells were washed and incubated with goat
anti-human
1gG(H+L) for 30 in at 4 'C. The washed cells were fixed and run on
FACSCelesta. FIGURE
17 depicts the mean fluorescence intensity (MF1) of the indicated chimeric
antibody as compared
to isotype and other negative controls and avelumab. Specific staining to CHO-
PD-L1 cells was
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seen for the PD-Li antibodies with very little non-specific binding to CHO
cells, even at high
antibody concentrations.
104211 The ability of chimeric PD-Li antibodies to modulate T cell function
was tested.
Monocyte-derived Dendritic Cells (moDC) were incubated with CellTrace Violet
(CTV)-labeled
allogeneic T cells in the presence of antibodies for 5 days. Proliferation was
measured by
FACSCelesta. Cytokine and cytolytic granules in supernatant were analyzed by
multiplex bead-
based assay. Each panel of FIGURES 18-20 shows two experiments: the left 5
bars show T
cells from donor 1 responding to moDC from donor 2, and the right 5 bars show
T cells from
donor 2 responding to moDC from donor 1.
104221 FIGURE 18 depicts the enhancement of T cell proliferation and cytokine
response to
allogeneic moDC in the presence of the indicated PD-Li antibodies compared to
isotype control
(001-1). FIGURE 18 shows CD4 T cell proliferation (FIGURE 18A), CD8 T cell
proliferation
(FIGURE 18B), TNEcc (FIGURE 18C), and 1FN-y levels (FIGURE I8D).
104231 FIGURE 19 depicts the enhancement of cytokine response to allogeneic
moDC in the
presence of indicated PD-Li antibodies compared to isotype control (001-1).
FIGURE 19
shows IL-2 (FIGURE 19A), IL-4 (FIGURE 19B), IL-6 (FIGURE 19C) and IL-10 levels
(FIGURE 19D).
104241 FIGURE 20 depicts the enhancement of degranulation in moDC-T cell MLR
in the
presence of the indicated PD-Li antibodies compared to isotype control (001-
1). FIGURE 20
shows soluble Fas Ligand (FIGURE 20A), Granzyme A (FIGURE 20B), perforin
(FIGURE
20C) and granulysin (FIGURE 20D).
104251 TABLE 15 is a summary of the biochemical and cellular activity of the
chimeric PD-
Li antibodies.
TABLE 15 Anti-PD-L1 chimeric IgG characterization summary
Characterizations 752- 767- 769- 771- 785- 788- Avelumab
hIgG1 hIgG1 hIgG1 hIgG1 hIgG1
hIgG1
Kd to hPD-L1, M 1.18x109 5.22x10-11 6.40x10-1 1.43x10' 1.64x10'
4.30x10' 5.53x10-1
Kd to cPD-L1, M 3.18x10-" 2.66x10' 5.50x10-1 1.12x10' 7.24x10'
8.67x101 9.94x10-1
IC50, nM (ELISA) 4.6 2 1.6 1.8 1.9 2.9
2
IC50, nM (Function) 1.5 0.6 0.5 1 0.5 0.9
0.5
Monomer% (SEC) 72% 97% 97% 100% 100% 100%
100%
Bind PD-L1- >100x >100x >100x >100x >100x
>100x >100x
CHO/CHO
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Characterizations 752- 767- 769- 771- 785- 788- Avelumab
hIgG1 hIgG1 hIgG1 hIgG1 hIgG1
hIgG1
PD-L2 binding No No No No No No
No
HCC827 Kd, nM 3.63 0.26 0.17 1.02 0.29 1.57
0.3
NCI-H292 Kd, nM 4.98 0.23 0.12 1.17 0.19 1.68
0.27
DC Int NS, 10.19 9.15 12.19 16.95 19.01
12.48 6.04
% lOnM
Donorl INS, 14.29 9.34 4.51 17.52 16.87
13.96 7.31
1nM
Pam, 0.94 1.13 1.34 6.85 3.44 -
3.44 -3.47
lOnM
DC Int NS, 29.19 18.05 20.17 24.5 22.37
28.07 21.33
cyo 1nM
Donor2 NS, 31.14 25.14 30.67 31.82 31.27
31.02 25.97
lOnM
Pam, 21.77 22.99 26.45 31.79 29.27
24.33 21.98
lOnM
Examnie
104261 This example describes the PD-Li antibody humanization. FIGURE 21A
depicts the
PAL769 VH sequence in mouse frameworks (769VH-wt; SEQ ID NO: 164) compared to
the
VH sequence in human frameworks (h769VH-mF0; SEQ ID NO: 199). CDRs identified
by
IMGT are shown in red (GFNIKDTY (SEQ ID NO: 161; IDPANDNT (SEQ ED NO: 162; and
AREGYGGSYGEGY (amino acids 97-109 of SEQ ID NO: 164). Note that CDRs provided
elsewhere in the application may be identified by other definitions (e.g.,
Kabat) and may vary.
FIGURE 21B depicts the PAL769 VL sequence in mouse frameworks (769Vk-wt; SEQ
ID NO:
167) compared to the VL sequence in human frameworks (h769Vk-mF0; SEQ ID NO:
242).
Highlighted amino acids in 769Vk-wt were back mutated and tested for activity
(data not
shown). A series of single back mutations (h769Vk-T531 (SEQ ID NO: 243);
h769Vk-A55F
(SEQ ID NO: 244); h769Vk-567Y (SEQ ID NO: 245); h769Vk-Y87F (SEQ ID NO: 246))
chosen for further study are also shown. FIGURE 21C depicts a series of 2 or 3
back mutations
as well as a potential deamidation motif on CDR-L3 (h769Vk-IY (SEQ ID NO:
247); h769Vk-
IF2 (SEQ ID NO: 248); h769Vk-tml (SEQ ID NO: 249); h769Vk-IF3 (SEQ ID NO:
200);
h769Vk-tm2 (SEQ ID NO: 201); h769Vk-tm3 (SEQ ID NO: 202)).
104271 Selected h769 mutations were produced and purified. FIGURE 22 depicts
UV traces
of size exclusion chromatographs of a selected group of humanized PD-Li
antibodies with
monomeric peaks at the expected retention times. As described supra, ForteBio
octet binding of
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purified humanized antibodies to recombinant hPD-Ll-his and cPD-Ll-his was
measured with
curves for hPD-L1 shown in FIGURE 23A and for cPD-L1 shown in FIGURE 23B. KD,
Kon
and Kdis values for human and cyno PD-Li are shown in TABLE 16.
TABLE 16
Human PD-Li
Cyno PD-Li
Clone ID KD (M) kon(l/Ms) kdis(1/s) KD (M)
kon(l/Ms) kdis(1/s)
769-wt 2.59x10"9 3.73x105 9.67x104" 2.92x10'9 2.38x105
6.85x10'
h769-IF3 2.64x10"9 3.30x105 8.71x10- 4 2.92x10"9 2.08x105 6.02x10'4
h769-tm2 2.48x1009 3.31x105 8.18x10-04 3.01x10-09 2.04x105 5.93x1004
h769-tm3 2.95x10'9 3.11x105 9.04x10- 4 3.11x10- 9 2.11x105 6.50x10'4
104281 Selected h769 antibodies were tested in the blocking ELISA as described
in Example 3
herein. Results are shown in FIGURE 24 and calculated ICsos (nM) are
indicated. Selected
h769 humanized PD-Li antibodies were characterized following removal of the
deamidation
motif in CDR-L3. FIGURE 25 depicts the ForteBio octet binding of purified
humanized
antibodies and recombinant hPD-Ll-his was measured. KD, Kon and Kdis values
for human
PD-Li are shown in TABLE 17. FIGURE 26 depicts UV traces of size exclusion
chromatographs of monomeric peaks at the expected retention times.
TABLE 17
Clone ID KD (M) kon(l/Ms) kdis(1/s)
h769-N93 1.98x10"9 2.92x105 5.80x10"4
h769-N93A 1.46x10"9 2.84x105 4.14x10"4
h769-N93T 1.82x10- 9 3.28x105 5.97x10'4
104291 FIGURE 27 shows that selected 769-hIgG1 humanized variants enhance T
cell response
to all ogenei c moDC. Avelumab was also tested in both an IgG1 format as well
as a IgG1
N297G format. PAL-767-1 was also used as a control (labeled blind in figures).
FIGURE 27
shows CD4 T cell proliferation (FIGURE 27A), Granzyme B (FIGURE 27B), and -MN-
1
(FIGURE 27C) as well as CD 8 T cell proliferation (FIGURE 27D), Granzyme A
(FIGURE
27E) and TNF'a levels (FIGURE 27F).
104301 FIGURE 28 shows that selected 769-hIgG1 humanized variants enhance T
cell response
to allogeneic moDC. FIGURE 28 shows Perforin (FIGURE 28A), soluble Fos (FIGURE
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28B), IL-6 (FIGURE 28C), Granulysin (FIGURE 28D), soluble Fas Ligand (FIGURE
28E)
and IL-10 levels (FIGURE 28F).
[0431] Selected 769-hIgG1 humanized variants were tested for their ability to
enhance PBMC
cytokine responses to CMV pp65. PBMCs were incubated with or without CMV pp65
protein
stimulation in the presence of antibodies for 4 days. Cytokine and cytolytic
granules in
supernatant were analyzed by multiplex bead-based assay.
[0432] FIGURES 29-33 show that selected 769-hIgG1 humanized variants enhance
PBMC
cytokine responses to CMV pp65, including by increasing levels of IL2, TNFct,
IL-6, IL-17A,
Granzyme A, Granulysin, and IFN-y. These results suggest that the 769-hIgG1
humanized
variants may be capable of enhancing an immune response in PBMCs.
Specifically, FIGURE
29 shows that selected 769-hIgG1 humanized variants enhance PBMC cytokine
responses to
CMV pp65. Levels of IL-2 (FIGURE 29A) and TNFcc (FIGURE 29B) are shown. FIGURE
30 shows that selected 769-hIgG1 humanized variants enhance PBMC cytokine
responses to
CMV pp65. Levels of IL-6 (FIGURE 30A) and IL-17A (FIGURE 30B) are shown.
FIGURE
31 shows that selected 769-hIgG1 humanized variants enhance PBMC cytokine
responses to
CMV pp65. Levels of Granzyme A (FIGURE 31A) and Granzyme B (FIGURE 31B) are
shown. FIGURE 32 shows that selected 769-hIgG1 humanized variants enhance PBMC
cytokine responses to CMV pp65. Levels of Perforin (FIGURE 32A) and Granulysin
(FIGURE 32B) are shown. FIGURE 33 shows that selected 769-hIgG1 humanized
variants
enhance PBMC IFN-y response to CMV pp65.
[0433] Next, an epitope binning sandwich assay was developed to determine
whether the
epitope of h769.T-1A overlapped with PAL-752, PAL-767, PAL-769, PAL-771, PAL-
785 or
PAL-788. h769.T-1A includes: the variable region h769.T (also referred to as
h769-N93T),
which includes a heavy chain variable region of SEQ ID NO: 199 and light chain
variable region
of SEQ ID NO: 204; and a human IgG1 constant region including a N297A
mutation. The
mouse IgG hybridoma version of each antibody was tested. The assay was
performed as
follows:
[0434] Step 1: 1st antibody of mouse IgGs was captured on AMC biosensor;
104351 Step 2: binding to hPD-L1-his antigen; and
[0436] Step 3: binding to h769.T-IA.
[0437] As shown in FIGURE 34, hPD-Ll-his that was bound to PAL752 (second to
top line)
and PAL788 (top line) can still bind to h769.T-1A, suggesting that these two
antibody epitopes
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do not overlap with the epitope of h769.T-1A, and that PAL767, PAL769, PAL771
and PAL785
have shared or overlapping epitopes with h769.T-1A.
Example 5
104381 This example describes the construction of PD-Li antibody sialidase
conjugates.
104391 An exemplary configuration of an anti-PD-Li antibody ASC is referred
to as -Janus,"
and contains one antibody arm (with one heavy chain and one light chain), and
one sialidase-Fc
fusion with a sialidase fused at the N-terminus of one arm of the Fc. Each Fc
domain
polypeptide in the Janus ASC contains either the "knob" (1366Y) or "hole"
(Y407T) mutation
for heterodimerizati on (residue numbers according to EU numbering, Kabat,
L.A., etal. (1991)
supra) (see, e.g., FIGURE 6B).
104401 A Janus PD-Li antibody sialidase conjugate was constructed using Neu2
with M1D,
V6Y, P62G, A93E, I187K, and C332A mutations, the variable region of anti-PD-Li
antibody
h769.T (as described in Example 4 herein, and also referred to as h769-N93T),
and a human
IgG1 Fc domain including an N297A mutation. This Janus PD-Li antibody
sialidase conjugate
(referred to as ASC1, and including a first polypeptide chain with amino acid
sequence SEQ ID
NO: 205, encoded by nucleotide sequence SEQ ID NO: 208, a second polypeptide
chain with
amino acid sequence SEQ ID NO: 206, encoded by nucleotide sequence SEQ ID NO:
209, and a
third polypeptide chain with amino acid sequence SEQ ID NO: 207, encoded by
nucleotide
sequence SEQ ID NO: 210) was expressed and characterized for purity using SDS-
PAGE and
enzymatic activity using 4MU-NeuAc as described below.
104411 ASC I was expressed in a 1,000 mL transfection of Expi293 human cells
using the
pCEP4 mammalian expression vector. The PD-Li antibody sialidase conjugate was
purified
using protein A followed by Ceramic Hydroxyapatite chromatography, quantified
with a UV-Vis
spectrophotometer (NanoDrop), and examined by SDS-PAGE as shown in FIGURE 35A.
ASC1 expressed well with an 89% purity after purification (FIGURE 35B).
104421 The activity of ASC1 was assayed by measuring the release of sialic
acid from the
fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-
NeuAc).
Specifically, an enzyme kinetics assay was performed by incubating a fixed
concentration of
enzyme at 1 nM with fluorogenic substrate 4MU-NeuAc at concentrations ranging
from 4000
p.1\4 to 7.8 M. ASC I was active with a Vmax of 5.5x107, causing the release
of sialic acid
which generated fluorescence. Assays were conducted at pH 5.6.
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104431 FIGURE 36 shows a binding assay (ForteBio octet binding) between
atezolizumab,
h769 hIgGl, h769-N93T or ASC1 and recombinant human PD-Li. TABLE 18 has the
binding
kinetics of the four test articles demonstrating very similar KDs in the 1-2
nM range.
TABLE 18
Clone KD (M) kon(l/Ms) kdis(1/s)
atezolizumab 1.61x10' 3.02x105 4.87x10- 4
h769 1.78x10-" 3.53x105 6.23x1e4
h769.T 176x10 9 3.57x105 6.29x10- 4
ASC1 1.72x10-09 4.56x105 7.66x10-04
104441 A second Janus PD-Li antibody sialidase conjugate was constructed using
Neu2 with
M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, and C332A mutations, the
variable
region of anti-PD-Li antibody h769.T (as described in Example 4 herein, and
also referred to as
h769-N93T), and a human IgG1 Fc domain including an N297A mutation. This Janus
PD-Li
antibody sialidase conjugate (referred to as ASC3, and including a first
polypeptide chain with
amino acid sequence SEQ ID NO: 205, encoded by nucleotide sequence SEQ ID NO:
208, a
second polypeptide chain with amino acid sequence SEQ ID NO: 213, encoded by
nucleotide
sequence SEQ ID NO: 215, and a third polypeptide chain with amino acid
sequence SEQ ID
NO: 214, encoded by nucleotide sequence SEQ ID NO: 216) was expressed and
characterized
for purity using SDS-PAGE and enzymatic activity using 4MU-NeuAc as described
below.
104451 ASC3 was expressed in a 2,000 mL transfection of Expi293 human cells
using the
pCEP4 mammalian expression vector. The PD-Li antibody sialidase conjugate was
purified
using protein A followed by cation exchange and Ceramic Hydroxyapatite
chromatography,
quantified with a UV-Vis spectrophotometer (NanoDrop), and examined by SDS-
PAGE. ASC3
expressed well with a 97% purity by SEC after purification (FIGURE 37A).
104461 The activity of ASC3 was assayed by measuring the release of sialic
acid from the
fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-
NeuAc).
Specifically, an enzyme kinetics assay was performed by incubating a fixed
concentration of
enzyme at 1 nM with fluorogenic substrate 4MU-NeuAc at concentrations ranging
from 4000
M to 7.8 M. Several batches of ASC3 was active with a Vmax of 1.15x108,
causing the
release of sialic acid which generated fluorescence as shown in FIGURE 37B.
Assays were
conducted at pH 5.6.
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104471 A series of additional PD-Li antibody sialidase conjugates were made. A
third was
constructed using Neu2 with M1D, V6Y, K9D, I187K, C332A, A93E, V363R, L365R,
E218A,
and C219N mutations, the variable region of anti-PD-Li antibody h769.T (as
described in
Example 4 herein, and also referred to as h769-N93T), and a human IgG1 Fc
domain including
an N297A mutation. This Janus PD-Li antibody sialidase conjugate (referred to
as ASC4 loss
of function (LOF), and including a first polypeptide chain with amino acid
sequence SEQ ID
NO: 205, encoded by nucleotide sequence SEQ ID NO: 208, a second polypeptide
chain with
amino acid sequence SEQ ID NO: 213, encoded by nucleotide sequence SEQ ID NO:
215, and a
third polypeptide chain with amino acid sequence SEQ ID NO: 217, encoded by
nucleotide
sequence SEQ ID NO: 218) was expressed and characterized for purity using SDS-
PAGE and
enzymatic activity using 4MU-NeuAc as described above.
104481 ASC4 LOF was expressed in a 1,000 mL transfection of Expi293 human
cells using the
pCEP4 mammalian expression vector. The PD-L1 antibody sialidase conjugate was
purified
using protein A followed by cation exchange and CHT Ceramic Hydroxyapatite
chromatography, quantified with a UV-Vis spectrophotometer (NanoDrop), and
examined by
SDS-PAGE. ASC4 LOF expressed well with a purity of 65% by SEC after
purification
(FIGURE 38). As expected, ASC4 LOF had no detectable activity using 4MU-NeuAc
as a
substrate.
104491 A fourth PD-Li antibody sialidase conjugate was constructed using Neu2
with MID,
V6Y, A42R, P62G, A93E, QI26Y, I187K, A242F, Q2701, and C332A mutations, the
variable
region of anti-PD-Li antibody h769.T (as described in Example 4 herein, and
also referred to as
h769-N93T), and a human IgG1 Fc domain including an N297A mutation. This Janus
PD-Li
antibody sialidase conjugate (referred to as ASC5, and including a first
polypeptide chain with
amino acid sequence SEQ ID NO: 205, encoded by nucleotide sequence SEQ ID NO:
208, a
second polypeptide chain with amino acid sequence SEQ ID NO: 213, encoded by
nucleotide
sequence SEQ ID NO: 215, and a third polypeptide chain with amino acid
sequence SEQ ID
NO: 219, encoded by nucleotide sequence SEQ ID NO: 220) was expressed and
characterized
for purity using SDS-PAGE and enzymatic activity using 4MU-NeuAc as described
above.
104501 ASC5 was expressed in a 1,000 mL transfection of Expi293 human cells
using the
pCEP4 mammalian expression vector. The PD-Li antibody sialidase conjugate was
purified
using protein A followed by cation exchange and Ceramic Hydroxyapatite
chromatography,
quantified with a UV-Vis spectrophotometer (NanoDrop), and examined by SDS-
PAGE. ASC5
expressed well with a purity of 98% monomeric heterodimer by SEC (FIGURE 39A).
ASC5
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was active with a Vmax of 1.4x108, causing the release of sialic acid which
generated
fluorescence as shown in FIGURE 39B.
104511 A fifth PD-Li antibody sialidase conjugates was constructed using Neu2
with M1D,
V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, and C332A mutations, the variable
region
of anti-PD-Li antibody h769.T (as described in Example 4 herein, and also
referred to as h769-
N93T), and a human IgG1 Fc domain including an N297A mutation. This Janus PD-
Li
antibody sialidase conjugate (referred to as ASC2, and including a first
polypeptide chain with
amino acid sequence SEQ ID NO: 205, encoded by nucleotide sequence SEQ ID NO:
208, a
second polypeptide chain with amino acid sequence SEQ ID NO: 206, encoded by
nucleotide
sequence SEQ ID NO: 209, and a third polypeptide chain with amino acid
sequence SEQ ID
NO: 211, encoded by nucleotide sequence SEQ ID NO: 212) was expressed and
characterized
for purity using SDS-PAGE and enzymatic activity using 4MU-NeuAc as described
above.
104521 ASC2 was expressed in a 1,000 mL transfection of Expi293 human cells
using the
pCEP4 mammalian expression vector. The PD-L1 antibody sialidase conjugate was
purified
using protein A followed by cation exchange and Ceramic Hydroxyapatite
chromatography,
quantified with a UV-Vis spectrophotometer (NanoDrop), and examined by SDS-
PAGE. ASC2
expressed well with a purity of 90% by SEC as shown in FIGURE 40A. ASC2 was
active with
a Vmax of 6.05x107, causing the release of sialic acid which generated
fluorescence. FIGURE
40B depicts the activity of ASC2 following protein A purification (ProA),
after cation exchange
(SP) and after Ceramic Hydroxyapatite chromatography (CHT). For comparison,
the same
results are also shown for ASC3. Both PD-Li antibody sialidase conjugates
demonstrated
improved activity as the molecules were purified to homogeneity.
104531 The ability of PD-Li antibody sialidase conjugates to bind human and
cynomolgus PD-
Li was confirmed. FIGURE 41 depicts human PD-L1 (FIGURE 41A) and cynomolgus PD-
L1
(FIGURE 41B) binding kinetics to selected of PD-Li antibody sialidase
conjugates as
compared to h769.T-1A (as described above in Example 4 herein). KD, Kon and
Kdis values for
human and cynomolgus PD-Li of the PD-Li antibody sialidase conjugates compared
to h769.T-
1A are shown in TABLE 19.
TABLE 19
hPD-L1 KD (M) kon(l/Ms) kdis(1/s) cPD-L1
KD (M) kon(l/Ms) kdis(1/s)
ASC3 1.73x10' 4.36x105 7.51x10" ASC3
2.26x10-" 3.67x105 8.21x104
ASC4 LOF 1.79x10' 4.49x105 7.55x10" ASC4 LOF 1.04x10' 4.23x10" 3 90x10"
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ASC5 1.66x10'9 4.33x105 7.21x10"4 ASC5 1.23x10'9 3.69x105
4.38x10"4
h769.T-1A 2.13x10'9 3.46x105 7.28x10"4 h769.T-1A 1.53x10"9 2.97x105 4.43x104
104541 The ability of PD-Li antibody sialidase conjugates to bind PD-Li on
HCC827 and
NCI-H292 cells was examined next. Cells were incubated with antibody (h769.T-
1A and
atezolizumab) and ASC3, ASC4 LOF, and ASC5 molecules at 4 C for 30 min. After
washing
with staining buffer, cells were incubated with AF647 Goat anti-human IgG(H+L)
in staining
buffer at 4 C for 30 min. After 2X wash with staining buffer, cells were
fixed and run on
FACSCelesta. FIGURE 42 depicts binding of PD-Li antibody sialidase conjugates
to HCC827
(FIGURE 42A) and NCI-H292 (FIGURE 42B) lung epithelial cell lines. The
apparent Kd
(nM) for each antibody is depicted in TABLE 20.
TABLE 20
Kd (nM) Kd (nM)
HCC827 NCI-H292
Atezolizumab 0.1127** 0.07495**
h769.T-1A 0.1249** 0.09732**
ASC3 0.2330* 0.1925
ASC4 LOF 0.5599* 0.4445
ASC5 0.4554* 0.3633
* = Calculated using data points up to 11 nM
** = Calculated using data points up to 3.7 nM
104551 The ability of PD-Li antibody sialidase conjugates to desialylate K562
and HT-29 cells
was examined. Cells were incubated with ASC5 and compared to ASC4 LOF at 37 C
overnight
(17 hours). HT-29 cells were lifted in Accutase at 37 C for 10 min. After wash
in staining
buffer, cells were incubated in biotin-PNA and live/dead in PBS at 4 C for 30
min. After
washing with PBS, cells were incubated with AF647-Strep in staining buffer at
4 C for 20 min.
Cells were washed twice with staining buffer and run immediately. FIGURE 43
depicts
desialylation by PD-Li antibody sialidase conjugates on K562 cells (FIGURE
43A) and HT-29
cells (FIGURE 43B).
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Example 6
[0456] This Example describes the in vivo administration of anti-PD-
Li antibody sialidase
conjugates (ASCs) containing human sialidases.
104571 Anti-PD-Li antibody sialidase conjugates were tested in a
transgenic mouse
engineered to express human PD-Li and human PD-1 in which mouse PD-Li and
mouse PD-1
have been disrupted (Biocytogen Inc.). Such double knock-in, knock-out mice
were injected
with a MC38 murine cancer cell line engineered to express human PD-Li. Mice, 6-
8 weeks of
age, were inoculated subcutaneously in the right lower flank region with
either the parent murine
cell line or human PD-Li expressing tumor cells for tumor development. Mice
were randomly
allocated to 4 groups of 8 animals each when tumors reached 50-100 mm3, mean ¨
75-100 mm3
and treated as shown in TABLE 21.
TABLE 21
Group Treatment Dose Route Schedule
1 001 - lA isotype control 10 mg/kg
(drug A)
2 ASC5 (drug B) 10 mg/kg Every
other day;
IP
8 doses
3 ASC4 LOF (drug C) 10 mg/kg
4 h769.T-1A (drug D) 5 mg/kg
[0458] Mice were treated via intraperitoneal injection of 10 mg/kg of
A SC5 or ASC4 LOF
(each as described above in Example 5 herein), 10 mg/kg of isotype control, or
5 mg/kg of
h769.T-1A (as described above in Example 4 herein), and tumor volume (mm3) was
recorded.
Mean tumor volumes for the individual mice for the indicated treatments were
determined.
[0459] As shown in FIGURE 44A, mice treated with ASC5 exhibit
statistically meaningful
reduced tumor volumes compared to mice treated with the control or ASC4 LOF PD-
Li
antibody sialidase conjugates. The reduced tumor volumes following treatment
with ASC5
relative to ASC4 LOF demonstrate the importance of the sialidase activity in
tumor reduction.
Tumor volumes for the individual mice for the indicated treatments are shown
in FIGURE 44B.
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Example 7
104601 This Example demonstrates the ability of anti-PD-Li antibody
sialidase conjugates
(ASCs) to block the PD-1 PD-Li interaction.
104611 Two lots of ASC5 (as described above in Example 5) were tested for
their ability to
block a biotinylated human PD-1 Fc fusion (hPD-1-Fc) from binding to human PD-
Li (hPD-
L1). ASC5 as well as atezolizumab and h769.T-1A (as described above in Example
4) were 3x
titrated and mixed with hPD-1-Fc at a final concentration of 1 ug/mL. The
mixture of antibody
and hPD-1-Fc was loaded on to hPD-L I coated ELISA wells for binding. ASCs or
antibodies
that bind the hPD-1 binding epitope on hPD-L I will compete for binding and
result in a
reduction of hPD-1-Fc binding signal. The residual binding of hPD-1-Fc to hPD-
L I was
detected with HRP conjugated streptavidin. The plate was developed with TMB
and Stop buffer
and the absorbance at 450 nm was read using a SpectraMax plate reader. A450
absorbance
curves and IC5os were generated using GraphPad Prism software. hPD-1-Fc only
(no antibody)
and buffer only (no antibody or hPD-1-Fc) were used as controls. As depicted
in FIGURE 45,
ASC5 blocked hPD-1-Fc binding to hPD-Li. IC5os for the two lots of ASC5 were
3.319 nM and
3.134 nM, which was slightly reduced relative to atezolizumab (IC50 of 1.305
nM) or h769.T-1A
(IC50 of 1.444 nM). It is contemplated that the difference in IC5ovalues was
due to the
difference between the antibody and ASC formats (e.g., ASC5 has only a single
PD-Li binding
site while atezolizumab and h769.T-1A each have two PD-L1 binding sites).
104621 ASC5 was also incubated with (i) engineered CHO-Kl cells expressing
human PD-Li
and TCR activating protein and (ii) Jurkat T cells expressing human PD-1, TCR
and a luciferase
reporter driven by an NEAT response element. Absent intervention, PD-Li
interacting with PD-
1 inhibits TCR-mediated luminescence, while blockade of the PD-L1/ PD-1
interaction results in
a luminescent signal. A luciferase substrate was added after 6 hours of
incubation and
luminescence was measured. Relative light units (RLU) were calculated by
subtracting
background (substrate and media only) from assay wells. Fold induction was
calculated by
dividing the RLU of induced cells minus background by the RLU of a no antibody
control minus
background (Fold induction = RLU (induced - background) / RLU (no antibody
control -
background)). Atezolizumab and h769.T-1A were used as positive controls, and
each produced
a fold induction of 4 to 5 over a range of 0.1 to 10 u.g/mL in this assay. As
shown in FIGURE
46, three different lots of ASC5 caused a dose dependent increase in
luminescence, indicating
that the ASC is capable of disrupting the PD-Li/PD-1 interaction. EC5os for
the three lots of
ASC5 were 11.54, 11.59 and 12.71 nM. EC5os for atezolizumab and h769.T-1A were
0.474 nM
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and 0.5596 nM, respectively. It is contemplated that the difference in EC50
values was due to the
difference between the antibody and ASC formats (e.g., ASC5 has only a single
PD-Li binding
site while atezolizumab and h769.T-1A each have two PD-Li binding sites).
Example 8
104631 This Example demonstrates the ability of anti-PD-Li antibody
sialidase conjugates
(ASCs) to remove sialic acid from the surface of human tumor cell lines and
primary immune
cells.
104641 Following incubation with ASCs, various cell types were
stained with a2,3 SiaFind
(Lectenz) and PNA lectin. Sialidase activity and removal of a2,3 sialic acid
linkages from the
cell surface results in decreased staining by ct2,3 SiaFind Sialidase activity
and exposure of the
underlying galactose sugar upon sialic acid cleavage results in increased
staining by PNA lectin.
104651 ASCs were tested on (i) BT-20, HT-29, and SK-BR-3 tumor cell
lines, (ii)
monocytic-derived dendritic cells (mDCs) generated from two separate healthy
donors by
treating isolated CD14+ monocytes with 50 ng/ml of both GM-CSF and IL-4, and
(iii) PBMCs
from two separate healthy donors thawed from frozen stocks. For mDCs and PBMCs
only, cells
were either stimulated with 300 ng/ml Pam3CSK4 or left unstimulated. For tumor
cells, no
stimulation was added. Cells were treated overnight (-15 hours) with prepared
1:3 serial
dilutions of ASC5 or ASC4 LOF (each as described above in Example 5), or
isotype control with
the highest concentration starting at 2,000 pg/ml. An 18-point curve was
generated for each cell
with each condition and each ASC or isotype concentration for each cell
condition was
completed in duplicate. After overnight treatment, tumor cells only were
treated with Accutase
for 15 minutes at 37 C to loosen cells off of the plate. All cells were
washed with PBS and
stained with Zombie Aqua cell viability kit at 1:1000 dilution in PBS on ice
for 15 minutes to
identify live cell populations. Subsequent cell washes using cell staining
buffer (Biolegend)
were completed between each blocking and staining step including after the
cell viability stain.
Additionally, staining and resuspension steps were also completed with cell
staining buffer.
Primary immune cells only were treated with Fe Receptor blocking agent FcX
(Biolegend) at
1:20 dilution on ice for 15 minutes. All cells were stained with a mixture of
PNA-AF647 (15
pg/m1) and SureLight488- a2,3 SiaFind (30 pg/ml; Lectenz) on ice for 30
minutes. Tumor cells
were resuspended and immediately read on BD FACS Celesta via BD Diva Software.
104661 Monocytic DCs were stained with BV421-CD11c and PE-DC-Sign
while PBMCs
were stained with PE-CD8, PercpCy5.5-CD56, BV421-CD14, BV650-CD19, and BV785-
CD3
(Biolegend) on ice for 30 minutes at a 1:40 dilution for all staining
antibodies (Biolegend).
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Primary immune cells were resuspended and immediately read on BD FACS Celesta
via BD
Diva Software. FloJo software was used to gate out non-debris, single, and
live cells.
Additionally, mDCs were gated as CD11c+/DC-Sign+ while PBMC populations were
separated
as CD56hi and CD56int NK cells, CD14hi and CD14int monocytic cells, and
CD3/CD8 T Cells.
The gMFI of alpha 2,3 SiaFind (Lectenz) and PNA for each population was put
into GraphPad
Prism software to generate IC50 (TABLE 22) and EC50 (TABLE 23) values,
respectively.
104671 ASC5 desialylated both tumor cells and primary human cells as
measured by a
reduction in a2,3 SiaFind staining, with IC5os between 10 and 100 ps/mL.
Following Pam3K
stimulation of the primary human cell populations, a clear reduction in IC5os
was observed in
mDCs from two different donors by 3 orders of magnitude as well as reduced
IC5os in CD14hi
and CD14int monocytes (TABLE 22). Pam3K stimulation was also shown to increase
PD-Li
expression in these cell types which would correlate with the reduced IC5os.
Increased PD-Li
expression leads to increased desialylation efficiency of ASC5.
104681 Likewise, ASC5 desialylated both tumor cells and primary human
cells as measured
by an increase in PNA staining, with EC5os between ¨100 and 1,000 [tg/mL.
Following Pam3K
stimulation of the primary human cell populations, a clear reduction in EC5os
was observed in
mDCs from two different donors (TABLE 23). Pam3K stimulation was also shown to
increase
PD-L1 expression in these cell types which appears to correlate with the
reduced EC5os.
Increased PD-L1 expression appears to lead to increased desialylation
efficiency of ASC5.
TABLE 22 - ICso Measured by a2,3 SiaFind-SL488 Staining
IC50
Cell Category
Cell No Stim Pam3K
Stim
(Donor if Applicable)
BT-20 90.26 NA
Tumor Cells (NA) HT-29 68.84 NA
SK-BR-3 52.53 NA
Differentiated Cells (Donor 1) mDCs 22.31
0.02999
Differentiated Cells (Donor 2) mDCs 17.81
0.02217
CD14hi Monocytes 19.35 3.468
CD14int Monocytes 14.82 1.223
PBMCs (Donor 3) CD56hi NK Cells 60.11 76.14
CD56int NK Cells 31.08 22.81
CD8 T Cells 51.09 63.62
CD14hi Monocytes 8.998 3.633
CD14int Monocytes 6.472 3.385
PBMCs (Donor 4) CD56hi NK Cells 10.92 5.329
CD56int NK Cells 13.24 10.17
CD8 T Cells 9.435 13.86
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TABLE 23 - ECso Measured by PNA-AF647 Staining
EC5o
Cell Category
Cell No Stim Pam3K
Stim
(Donor if Applicable)
BT-20 1769 NA
Tumor Cells (NA) HT-29 1102 NA
SK-BR-3 1549 NA
Differentiated Cells (Donor 1) mDCs 2353 511.1
Differentiated Cells (Donor 2) mDCs 79557 5847
CD14hi Monocytes 86.15 91.12
CD14int Monocytes 1435 3612
PBMCs (Donor 3) CD56hi NK Cells 248.3 542.8
CD56int NK Cells 257.8 252.9
CD8 T Cells 617 628.2
CD14hi Monocytes 168.3 162
CD14int Monocytes 1212 2272
PBMCs (Donor 4) CD56hi NK Cells 225.9 229
CD56int NK Cells 243 603.6
CD8 T Cells 917.2 905.3
Example 9
104691 This Example demonstrates the impact of anti-PD-Li antibody sialidase
conjugates
(ASCs) on cytokine release in a human dendritic cell and T cell coculture
experiment.
104701 CD14+ monocyte-derived dendritic cells were generated by a 6-day
culture in GM-CSF
and IL-4 (50 ng/ml each) and co-incubated with allogeneic T cells at 1:2 DC:T
ratio in the
presence of test articles for 3 days. Supernatants were collected for cytokine
analysis by
LEGENDplex 13-plex panel. Each data point represents a separate DC-T donor
pair (for each
test condition two independent experiments were conducted that each included
four replicates).
ASC5 (as described above in Example 5) was used at 700 nM (100 mg/mL), h769.T-
1A (as
described above in Example 4) and atezolizumab were used at 70 nM (10 mg/mL),
and isotype
control was used at 100 mg/mL. FIGURE 47A depicts the fold change in IL-2
following
treatment with ASC5, h769.T-1A, and atezolizumab compared to isotype control.
FIGURE
47B, FIGURE 47C, and FIGURE 47D show similar data for IFN-y, IL-8 and MCP1,
respectively. All four cytokines increased following ASC5 treatment by more
than 2 fold, and
the increase was at least as much as following treatment with h769.T-1A or
atezolizumab.
Example 10
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104711 This Example describes the in vivo administration of anti-PD-
Li antibody sialidase
conjugates (ASCs) containing human sialidases.
104721 ASC5 (as described above in Example 5) was tested in a
transgenic C57BL6 mouse
engineered to express human PD-Li and human PD-1 in which mouse PD-Li and
mouse PD-1
have been disrupted (Biocytogen Inc.). Mice were injected with a MC38 murine
cancer cell line
engineered to express human PD-Li. Mice, 6-9 weeks of age, were inoculated
subcutaneously
in the right lower flank region with tumor cells for tumor development Mice
were randomly
allocated to groups of 8 animals each when tumors reached 90-136 mm3, mean ¨
109 mm3
104731 Mice were treated via intraperitoneal injection of ASC5 at
either 1, 3, 10, or 30
mg/kg, atezolizumab at 0.5 or 5 mg/kg, h769.T-1A (as described above in
Example 4) at 5
mg/kg, or isotype control at 30 mg/kg, and tumor volume (mm3) was recorded.
FIGURE 48A
shows tumor growth through Day 18. FIGURE 48B is an analysis of the Day 18
data,
demonstrating a significant reduction in tumor growth upon administration of
ASC5 at 30
mg/kg, comparable to the response of atezolizumab and h769.T-1A at 5 mg/kg.
TABLE 24
depicts tumor growth inhibition (TGITv) calculated at day 18 for each
treatment. TGITv ¨ I1-
(TVtest group ¨ TVtest group at day()) (TVcontrol group ¨ TVcontrol group at
day 0)} X 100%.
TABLE 24
Treatment TGIry
0.5 mg/kg atezolizumab 11.1%
5 mg/kg atezolizumab 71.8%
5 mg/kg h769.T-1A 82.1%
1 mg/kg ASC5 5.7%
3 mg/kg ASC5 21.4%
10 mg/kg ASC5 11.1%
30 mg/kg ASC5 64.4%
104741 A CT26 mouse tumor line engineered to express human PD-Li was
grown as a
syngeneic subcutaneous tumor in a transgenic BALB/c mouse engineered to
express human PD-
Li and human PD-1 and in which mouse PD-Li and mouse PD-1 have been disrupted
(Gempharmatech Inc.). Mice, 8-9 weeks of age, were inoculated subcutaneously
in the right
lower flank region with tumor cells for tumor development. Mice were randomly
allocated to
three groups of six animals each when tumors reached 90-120 mm3, with a group
mean of
104.06-104.36 mm3.
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104751 Mice were treated via intraperitoneal injection of ASC5 (as
described above in
Example 5; 10 mg/kg), h769.T-1A (as described above in Example 4; 5 mg/kg), or
isotype
control (10 mg/kg), and tumor volume (mm3) was recorded. FIGURE 49A shows
percent
tumor growth inhibition (TGI) through Day 18. FIGURE 49B is an analysis of the
Day 18 data,
demonstrating significant reduction in tumor growth upon administration of
ASC5, which was
Greater than the reduction for h769.T-1A.
104761 A dose response experiment with ASC5 was carried out in the
CT26 mouse model.
Mice were treated via intraperitoneal injection at 3, 10 and 30 mg/kg of ASC5,
10 mg/kg ASC4
(LOF), 5 mg/kg atezolizumab, and 30 mg/kg isotype control. 6 mice per group (7-
9 weeks of
age at inoculation) were randomized when tumors reached 76-125 mm3 (group mean
102-103
mm3). The humane endpoint was at 3,000 mm3 tumor volume. FIGURE 50A shows
tumor
growth inhibition (TGI) through Day 16. FIGURE 50B is an analysis of the Day
16 data
demonstrating significant dose dependent reduction in tumor growth upon
administration of
ASC5.
INCORPORATION BY REFERENCE
104771 The entire disclosure of each of the patent and scientific documents
referred to herein is
incorporated by reference for all purposes.
EQUIVALENTS
104781 The invention may be embodied in other specific forms without departing
from the
spirit or essential characteristics thereof The foregoing embodiments are
therefore to be
considered in all respects illustrative rather than limiting on the invention
described herein.
Scope of the invention is thus indicated by the appended claims rather than by
the foregoing
description, and all changes that come within the meaning and range of
equivalency of the
claims are intended to be embraced therein.
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SEQUENCE LISTING
104791 SEQ ID NO: 1:
MASLPVLQKE SVFQS GAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAPTH
QVQWQAQEVVAQARLDGHRSMNPC PLYDAQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHP I QRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGS CAYSDLQSMGTGPDCS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYL PQ
104801 SEQ ID NO: 2:
ME DLRPMAT C PVLQKE T L FRT GVHAYR I PALLYLKKQKT LLAFAEKRAS KT DEHAE L I
VLRRGS
YNEATNRVKWQ PEEVVT QAQLEGHRSMNP C PLYDKQTKT L FL FFIAVPGRVSEHHQLHTKVNVT
RLCCVSSTDHGRTWSP I QDL TETT I GS THQEWAT FAVGPGHCLQLRNPAGSLLVPAYAYRKLHP
AQKPTPFAFC F I S LDHGHTWKLGNFVAENS LE CQVAEVG T GAQRMVYLNARS FLGARVQAQS PN
DGLDFQDNRVVSKLVEPPHGCHGSVVAFFINP I SKPHALDTWLLYTHPTDSRNRTNLGVYLNQMP
L DP TAWSE P T L LAMGI CAYS DLQNMGQGPDGS PQFGCLYE SGNYEE I I FL I FTLKQAFP
TVFDA
104811 SEQ ID NO: 3:
EDLRP
104821 SEQ ID NO: 4:
MEDLRP
104831 SEQ ID NO: 5:
DKTHT C PPC PAPELLGGP SVFL FP PKPKDTLMI SRT PEVT CVVVDVSHE DPEVKFNWYVDGVEV
HNAKTKPREE QYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAP I E KT I SKAKGQ PRE PQV
YTLPP SREEMTKNQVS L T CLVKGFYP S D IAVEWE SNGQPENNYKT TP PVLDS DGS FEL Y SKL
TV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
104841 SEQ ID NO: 6:
ACAGT GGAAAAGT CC G T GGTGTT CAAGGC C GAGGGC GAG CAC T T CAC C
GACCAGAAAGGCAATA
C CAT CGTCGGC TCTGGCAGCGCCGGCACCACCAACTACT T TAGAATCC C CGCCAT GT GCACCAC
CAGCA_AGGGCACCAT T GT GG T GT T CGCCGACGCCAGACACAACACCGC CAGCGAT CAGAGCT IC
AT CGATACCGC T GCCGC CAGATCTACCGAT GGC GGCAAGACC T GGAACAAGAAGAT CGC CAT C T
ACAAC GACCGC GT GAACAGCAAGC TGAGCAGAGT GAT GGACCC TACC T GCATCGTGGCCAACAT
C CAGGGCAGAGAAACCAT CC TGGICATGGICGGAAAGIGGAACAACAACGATAAGACC T GGGGC
GCCTACAGAGACAAGGCCCC TGATACCGAT T GGGACC TCGT GC T GTACAAGAGCACCGAT GAC G
GCGT GACC T T CAGCAAGGT GGAAACAAACAT C CAC GACAT CGT GACCAAGAACGGCAC CAT C T C
T GCCATGCTCGGCGGCGT T GGATCTGGCC T GCAAC T GAT GAT GGCAAGC T GGT GI TCCCCGTG
CAGAT GGT CC GAACAAAGAATAT CAC CAC CGT GC T GAATACCAGCT T CAT C TACAGCAC CGACG
GCATCACATGGTCCCTGCCTAGCGGCTAC T GT GAAGGCT T TGGCAGCGAGAACAACATCATCGA
G T T CAACGC CAGC C T GG T CAACAACAT CC GGAAC.AGCGGCC T GC GGAGAAGC T T
CGAGACAAAG
GAC T T C GGAAAGACG T GGAC C GAG TTTCCTC CAAT GGACAAGAAGGT GGACAAC C GGAAC CAC
G
GCGTGCAGGGCAGCACAA.TCACAATCCCTAGCGGCAACAAACTGGIGGCCGCTCACTC TAGCGC
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C CAGAACAAGAACAAC GAC TACAC CAGAAGC GACAT CAG CCTG TACGC C CACAAC C T G TACAGC
GGCGAAGTGAAGCTGATCGACGACTTCTACCCCAAAGTGGGCAATGCCAGCGGAGCCGGCTACA
GC T GT C TGAGC TACCGGAAAAATGTGGACAAAGAAACCC TGTACGTGGT GTACGAGGCCAACGG
CAG CAT C GAG T T T CAGGAC C T GAG CAGACAT C T GCCC GT GAT CAAGAGC TACAAC
104851 SEQ ID NO: 7:
END FGLVQPLVTMEQLLWVS GRQ I GSVDT FRI PL I TAT PRGT LLAFAEARKMS S S DE GAKFIAL
RRSMDQGS TWS PTAFIVNDGDVPDGLNLGAVVSDVE TGVVFL FYSLCAHKAGCQVAS TMLVWSK
DDGVSWS T PRNL S LD I GTEVFAPGPGS G I QKQREPRKGRL IVCGHGT LE RDGVFCLL S DDHGAS
WRYGS GVS G I PYGQPKQENDFNPDECQPYELPDGSVVINARNQNNYHCHCRIVLRSYDACDTLR
PRDVT FDPE LVDPVVAAGAVVT S S G IVFFSNPAHPE FRVNL TLRWS FS NGT SWRKE TVQLWPGP
S GYS S LAT LE G SMDGEE QAP QLYVLYEKGRNHY TE S I S VAK I SV
104861 SEQ ID NO: 8:
MEEVT TCS ENS PL FRQEDDRG I TYRI PALLY' PPTHT FLAFAEKRS TRRDE DALHLVLRRGLR I
GQLVQWGPLKPLMEATLPGHRTMNPCPVWEQKSGCVFLFF I CVRGHVTERQQ IVSGRNAARLCF
I YS QDAGCSWSEVRDL TEEVI GS E LKHWAT FAVGPGHG I QLQSGRLVI PAY T YY IPSWF FC
FQL
P CKTRPHS LM I YSDDLGVTWHHGRL I RPMVTVE CEVAEVT GRAGHPVL YC SART PNRCRAEAL S
T DHGEGFQRLALSRQLCEPPHGCQGSVVS FRPLE I PHRCQDS S SKDAP T I QQSSPGS S LRLEEE
AGT P S E SWLL Y S HP T S RKQRVDL G I YLNQ T PLEAACWSRPW I
LHCGPCGYSDLAALEEEGLFGC
L FE CGTKQE CE Q IAFRL FT HRE I LSHLQGDCT S PGRNPS Q FKSN
104871 SEQ ID NO: 9:
MRPADL PPRPMEE S PAS S SAP TE TEE PGS SAEVMEEVI C S FNS PL FRQE DDRG I TYR'
PALLY
I PPTHT FLAFAEKRS TRRDE DALHLVLRRGLR I GQLVQWGPLKPLMEAT LPGHRTMNPCPVWEQ
KS GCVFL FF I CVRGHVTERQQ IVS GRNAARLC F I YS QDAGCSWSEVRDL TEEVI GS E LKHWAT
F
AVGPGHG I QLQSGRLVI PAY T YY I PSW FFG FQL PCKTRPH S LM I YSDDLGVTWHHGRL I
RPMVT
VE CEVAEVT GRAGHPVLYC SART PNRCRAEAL S DHGE G FQRLALS RQL CE P PHGCQG SVVS FR
P LE I PHRCQDS S SKDAPT I QQSSPGS SLRLEEEAGT PSE SWLLYSHPT S RKQRVDL G I YLNQT
P
LEAACWSRPW I LHCGPCGYS DLAALEEEGL FGCL FE CGT KQE CE Q IAFRL FTHRE I LSHLQGDC
T S PGRNPS QFKSN
104881 SEQ ID NO: 10:
MGVPRTPSRTVL FERERTGL TYRVPSLLPVPPGPTLLAFVEQRLSPDDSHAHRLVLRRGTLAGG
SVRWGALHVLGTAALAEHRSMNPCPVHDAGTGTVFL FFIAVLGHTPEAVQ IATGRNAARLCCVA
SRDAGLSWGSARDLTEEAI GGAVQDWAT FAVGPGHGVQL P SGRLLVPAYTYRVDRREC FGKI CR
T S PHS FAFYS DDHGRT WRCGGLVPNLRS GE CQLAAVDGGQAGS FLYCNARS PLGSRVQALS T DE
GT S FL PAERVAS L PE TAWGC QGS IVGFPAPAPNRPRDDSWSVGPGSPLQPPLLGPGVHE PPEEA
AVDPRGGQVPGGP FS RLQPRGDGPRQPGPRPGVS GDVGS WT LAL PMP FAAPPQSPTWLLYSHPV
GRRARLHMG I RLS QS PLDPRSWTE PWVI YE GP S GYS DLAS I GPAPEGGLVFACLYES GARTSYD
E IS FCT FS LREVLENVPAS PKPPNLGDKPRGCCWPS
104891 SEQ ID NO: 11:
MMS SAAFPRWL SMGVPRT PSRTVL FERERTGL TYRVPSLL PVPPGPTLLAFVEQRLS PDDSHAH
RLVLRRGTLAGGSVRWGALHVLGTAALAEHRSMNPCPVHDAGTGTVFL FF IAVLGHT PEAVQ IA
T GRNAARLCCVASRDAGLSWGSARDL TEEAI GGAVQDWAT FAVGPGHGVQL P S GRLLVPAYT YR
VDRRE C FGK I CRT SPHS FAFY S DDHGRTWRCGGLVPNLRS GE CQLAAVDGGQAGS FLYCNARS
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LGSRVQALS TDEGTSFLPAERVASLPETAWGCQGS IVGFPAPAPNRPRDDSWSVGPGS PLQPPL
LGPGVHEPPEEAAVDPRGGQVPGGPFSRLQPRGDGPRQPGPRPGVSGDVGSWTLALPMP FAAPP
QS P TWLLYS HPVGRRARLHMG IRL SQS PLDPRSWTE PWVI YEGPSGYS DLAS IGPAPEGGLVFA
CLYESGARTSYDE ISFCTFSLREVLENVPASPKPPNLGDKPRGCCJPS
[0490] SEQ ID NO: 12:
MAS LP
[0491] SEQ ID NO: 13:
AS LP
[0492] SEQ ID NO: 14:
TVEKSVVF
[0493] SEQ ID NO: 15:
GDYDAPTHQVQW
104941 SEQ ID NO: 16:
SMDQGSTW
[0495] SEQ ID NO: 17:
S TDGGKTW
[0496] SEQ ID NO: 18:
PRPPAPEA
104971 SEQ ID NO: 19:
QTPLEAAC
[0498] SEQ ID NO: 20:
NPRPPAPEA
[0499] SEQ ID NO: 21:
S QNDGES
[0500] SEQ ID NO: 22:
LSHSLST
[0501] SEQ ID NO: 23:
GAGAACGACT T TGGACIGGTGCAGCCICTGGICACCATGGAACAGCTGCTGTGGGTTTCCGGCA
GACAGATCGGCAGCGTGGACACCTICAGAATCCCICTGATCACCGCCACACCTAGAGGCACCCT
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GC T GGCCT T T GCCGAGGCCAGAAAGAT GAGCAGC TC TGACGAGGGCGCCAAGTT TAT T GCCCTG
AGGCGGTC TAT GGACCAGGGC TCTACAT GGTCCCC TACCGCC T TCATCGT GAACGAT GGCGACG
TGCCCGATGGCCTGAATCTGGGAGCTGTGGTGTCCGATGTGGAAACCGGCGTGGTGTTCCTGTT
C TACAGCC T GT GT GCCCACAAGGCCGGT T GTCAGGT GGCCAGCACAAT GC TCGT GT GGT CCAAG
GACGACGGCGT GTCCTGGT C TACCCC TAGAAACCIGAGCC T GGACATCGGCACCGAAGT GT T TG
C TCCAGGACC T GGC T C I GGCAT CCAGAAGCAGAGAGAGC CCAGAAAGGGCAGAC T GAT C GTGT G
T GGCCACGGCACCCIT GAGAGAGATGGCGT I I TC T GCCT GC T GAGCGAC GAT CAT GGC GCCTC
T
T GGAGATACGGCAGCGGAGT GICT GGAATCCCT TAC GGC CAGCC TAAGCAAGAGAAC GAT TT CA
ACCCCGACGAGTGCCAGCCT TACGAGC T GCC T GAT GGCAGCGTCGTGAT CAACGCCCGGAACCA
GAACAACTACCAC TGCCAC T GCCGGATCGT GC T GAGAAGC TACGACGCC T GCGATACCC T GCGG
CC TAGAGAT GT GACCT TCGATCCT GAGC T GGT GGACCCT GT T GT TGCCGC T GGT GCCGT CGT
GA
CATC TAGCGGCAT CGT GI T C T TCAGCAAC CC T GC T CACC CCGAGT TCAGAGT GAAT CT
GACCC T
GCGGTGGTCCT TCAGCAAT GGCACAAGC T GGCGGAAAGAAACCGTGCAGC T T TGGCC T GGACC T
AGCGGCTACTCTTCTCIGGCTACACTGGAAGGCAGCATGGACGGCGAAGAACAGGCCCCTCAGC
T GTACGTGC T GTACGAGAAGGGCAGAAAC CAC TACACCGAGAGCAT CAGCGT GGCCAAGAT CA G
CGTT
105021 SEQ ID NO: 24:
AT GGCCAGCC T GCCTGT GC T GCAGAAAGAAAGCGIGTICCAGTC TGGCGCCCACGCC TACAGAA
T TCCCGCTC T GC T GTATC T GCCAGGCCAGCAGICIC TGC T GGC T TTCGC T GAACAGCGGGCCAG
CAAGAAGGAT GAGCAC GC C GAAC T GAT CGT GC T GC GGAGAGGC GAT TAC GAC GC C C C
TACACAT
CAGGT GCAGT GGCAGGC T CAAGAGGT GGT GGC T CAGGCTAGAC T GGAC GGCCACAGAT C TAT GA
ACCCCTGTCCTCTGTACGATGCCCAGACCGGCACACTGT T TC T GT TC T T TATCGC TAT CCCCGG
CCAAGTGACCGAGCAGCAGCAGCTGCAGACAAGAGCCAACGTGACCAGACTGTGTCAAGTGACC
TCCACCGACCACGGCAGAACCIGGICTAGCCCTAGAGATCTGACCGACGCCGCCATCGGACCTG
CC TATAGAGAGT GGTCCACC T TCGCCGTIGGACCIGGACACTGTCTCCAGCTGCACGACAGGGC
TAGATC IC I GG T GGTGCC T GCCTACGCC TATAGAAAGCT GCACCCCAT C CAGCGGCC TAT TCC T
AGCGCC TIC T GC T TIC T GAGCCACGATCACGGCAGGACAT GGGCCAGAGGACAT T TCGT GGCCC
AGGACACAC T GGAATGCCAGGIGGCCGAAGIGGAAACCGGCGAGCAGAGAGTCGT GACCC TGAA
C GC CAGAT C T CAC C T GAGAG C CAGAG T GCAGGCCCAGAGCACAAACGAC GGCCT GGAT T T
CCAA
GAGAGCCAGCTGGICAAGAAACTGGTGGAACCICCICCACAGGGCTGTCAGGGAAGCGTGATCA
GC T T TCCATC T CC TAGAAGCGGCCCT GGC TC TCC T GC TCAGT GGCTGC T
GTATACACACCCCAC
ACACAGCT GGCAGAGAGCCGATCT GGGCGCC TACC T GAATCC TAGACC T CC T GCTCC T GAGGC T
T GGAGCGAAC C T CT TC T GC T GGCCAAGGGCAGC T GI GCC TACAGCGAT C T GCAGTC TAT
GGGCA
CAGGCCCT GAT GGCAGCCC T C TGT IT GGC T GTC T GTACGAGGCCAACGAC TACGAAGAGATCGT
GT TCC TGAT GT TCACCC T GAAGCAGGCC T T TCCAGCCGAGTACC TGCC T CAA
[0503] SEQ ID NO: 25:
AT GGAGGAAGT GACCACC T GTAGC T TCAACAGCCC TC T GT TCCGGCAAGAGGACGACCGGGGCA
T CACC TACAGAAT CCC T GC T C TGC T GTACAT CCC T CC TACACACACC T T
TCTGGCCTTCGCCGA
GAAGC GGAGCAC CAGAC GAGAT GAAGAT GCCC T GC ACCT GGT GC TGAGAAGAGGCC T GAGAAT C
GGACAGCTGGTGCAGIGGGGACCTCTGAAGCCTCTGATGGAAGCCACACTGCCCGGCCACAGAA
CCATGAATCCTTGICCIGTGTGGGAGCAGAAAAGCGGCTGCGTGITCCTGTTCTICATCTGCGT
GCGGGGCCACGTGACCGAGAGACAGCAAATCGTGTCCGGCAGAAACGCCGCCAGACTGTGCTTC
ATCTACAGCCAGGATGCCGGCTGCTCTTGGAGCGAAGTTCGGGATCTGACCGAAGAAGTGATCG
GCAGCGAGCTGAAGCACTGGGCCACATTTGCTGTTGGCCCTGGCCACGGAATCCAGCTGCAATC
TGGCAGACTGGTCATCCCCGCCTACACCTACTATATCCCCAGCTGGTTCTTCTGCTTCCAACTG
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CC T T GCAAGACCCGGCC TCACAGCC T GAT GATC TACAGCGACGATCT GGGCGTGACAT GGCACC
ACGGCAGAC T GAT CAGACCCATGGTCACC GT GGAAT GCGAGGT GGCCGAAGT GACAGGCAGAGC
T GGACACCC T GT GCTGTAC T GCTC TGCCAGAACACCCAACCGGT GTAGAGCCGAGGC T C T GTC T
ACAGATCACGGCGAGGGCT T TCAGAGAC T GGCCC TC TCTAGACAGCT GT GCGAACC TCC TCAT G
GCIGTCAGGGCAGCGIGGTGICCTICAGA_CCTCTGGAAATCCCTCACCGGTGCCAGGAC_AGCAG
C T C TAAGGAT G C C CC TAC CAT CCAGCAGT C TAGCCC TGGCAGCAGCC T GAGACT
GGAAGAGGAA
GCCGGAACAC C TAGCGAGAGC T GGCT GC T GTAC T C T CAC C C CAC CAGCAGAAAGCAGAGAG T
GG
ACC T GGGCAT C TACCT GAAT CAGACCCC T C T GGAAGCCGCC T GT TGGAGCAGACC T T GGAT
TC T
GCAC T GTGGCCC T TGCGGC TACTC T GATC T GGCCGC TCT GGAAGAAGAGGGCCTGT TCGGCT GC
C T GT T TGAGT GCGGCACAAAGCAAGAGT GCGAGCAGATCGCC T TCCGGC T GT TCACCCACAGAG
AGAT CC T GAG C CAT C T GCAGGGCGAC T GCACAAGCCCAGGCAGAAAT CCCAGCCAGT T CAAGAG
CAAC
105041 SEQ ID NO: 26:
AT GGGCGT GC C CAGAACACC CAGCAGAAC CGT GC I GI IC GAGAGAGAGAGGACCGGCC T GACCI
ACAGAGTGCCT TC TCT GC T GCCT GTGCC TCC T GGACC TACAC T GCTGGCC T TCGT
GGAACAGAG
AC T GAGCCCC GAT GAT IC T CACGCCCACAGAC I GGT GCT GAGAAGAGGAACACT GGC T GGCCGC
T CT GT TAGA T GGGGAGCAC T GCAT GT GCT GGGCACAGCT GC TC T TGCC GAGCACAGAT C
CAT GA
ATCCC TGTCC T GT GCACGACGCCGGAACCGGCACAGTGT T TC T GT TC T T TATCGCCGT GC TGGG
CCACACACCTGAGGCCGITCAAATTGCCACCGGCAGAAAIGCCGCCAGACTGIGITGTGTGGCC
I CCAGAGAIGCCGGCCIGT C I IGGGGATC I GCCAGAGAT C I GACCGAGGAAGCCAT IGGCGGAG
CCGTTCAGGAT TGGGCCACAT IT GCT GT I GGACC T GGACACGGCGTGCAGC T GCCAAG T GGTAG
AC T GC TGGT GCCIGCCIACACATACAGAGIGGATCGGAGAGAGT GCT T CGGAAAGATC T GCCGG
ACAAGCCCTCACAGCTTCGCCTTCTACTCCGACGATCACGGCCGGACT T GGAGAT GT GGT GGCC
T GGT GCCTAAT CI GAGAAGC GGCGAAT GT CAC I GGCCGCCGT I GAT GGT GGACAGGC T GGCAG
CTTCCIGTACTGCAACGCCAGATCTCCTCTGGGCTCTAGAGTGCAGGCCCTGICTACCGATGAG
GGCACCAGTITTCTGCCCGCCGAAAGAGTTGCCICTCTGCCTGAAACA_GCCTGGGGCTGICAGG
GCTCTATCGTGGGATTICCTGCTCCTGCTCCAAACAGACCCCGGGACGATTCTIGGAGTGTCGG
CCC T GGATC T CCACTGCAGCC TCCAT T GC T T GGACCAGGCGT TCACGAGCCACCT GAAGAGGC T
GCCGT TGATCC TAGAGGCGGACAAGT I CC I GGCGGCCCT T T TAGCAGAC T GCAGCCAAGAGGCG
ACGGCCCTAGACAACC T GGACCAAGACC T GGCGTCAGCGGAGAT GT T GGC TC T T GGACAC TGGC
CCTGCCTATGCCTTTTGCCGCTCCTCCTCAGTCTCCTACCTGGCTGCTGTACTCTCACCCTGTT
GGCAGACGGGCCAGACTGCACATGGGCATCAGACTGTCTCAGAGCCCTCTGGACCCCAGAAGCT
GGACAGAGCCT T GGGT CAT C TAT GAGGGC CC TAGCGGCTACAGCGAT CT GGCCTC TAT TGGCCC
AGC T CC TGAAGGCGGAC T GG T GT T CGC T T GTC T GTATGAGAGCGGCGC CAGAACCAGC
TACGAC
GAGATCAGCT TCIGCACCT T CAGCCIGCGCGAGGIGC GGAAAAIGIGCCCGCCICICC TAAGC
C TCC TAACC T GGGCGATAAGCCTAGAGGC T GT T GC T GGCCATC T
105051 SEQ ID NO: 27:
MT GERPS TAL PDRRWGPRI LGFWGGCRVWVFAAI FLLLSLAASWSKA
105061 SEQ ID NO: 28:
MDMRVPAQLLGLLLLWLPGARC
105071 SEQ ID NO: 29:
YGTL
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[0508] SEQ ID NO: 30:
MTVEKSVVFKAEGEHFIDQKGNT IVGS GS GGTTKY FRI PAMCT TSKGT IVVFADARHNTASDQS
F DTAAARS TDC-IGKIWNKKIAIYNDRVNSKLSRVMDPTC IVAN I OGRE T LVMVGKWNNNDKTW
GAYRDKAPDTDWDLVLYKS T DDGVT FSKVE TNI HD IVTKNGT I SAMLGGVGSGLQLNDGKLVFP
VQMVRTKNI T TVLNTS FIYS TDGI TWSLPSGYCEGFGSENNI IE FNAS LVNNIRNS GLRRS FE T
KDFGKTWTE FP PMDKKVDNRNHGVQGS TITI PS GNKLVAAHS SAQNKNNDYTRS DI S LYAHNLY
S GEVKL DD FY PKVGNA S GAGYS CLS YRKNVDKE T LYVVYEANGS IPQDLSRHLPVIKSYN
[0509] SEQ ID NO: 31:
E PKSCDKTHTC P PCPAPELL GGPSVFL FP PKPKDTLMI S RT PEVTCVVVDVSHEDPEVK FNWYV
DGVEVHNAKT KPREEQYNS T YRVVSVL TVLHQDWLNGKE YKCKVSNKAL PAP I EKT I SKAKGQP
RE PQVYTL P P SREEMTKNQVS LTCLVKGFYP S D IAVEWE SNGQPENNYKT TPPVLDS DGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0510] SEQ ID NO: 32:
DKTHT CPPCPAPELLGGP SVFL FP PKPKDTLMI SRT PEVT CVVVDVSHE DPEVKFNWYVDGVEV
HNAKTKPREE QYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAP I E KT I SKAKGQ PRE PQV
YTLPP SREEMTKNQVS L T CLVKGFYP S D IAVEWE SNGQPENNYKT TP PVLDS DGS FEL T SKL
TV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0511] SEQ ID NO: 33:
E PKS CDKTHT C P PCPAPELL GGPSVFL FP PKPKDT LMI S RT PEVTCVVVDVSHEDPEVK FNWYV
DGVEVHNAKT KPREEQYNS T YRVVSVL TVLHQDWLNGKE YKCKVSNKAL PAP I EKT I SKAKGQP
RE PQVYTL P P SREEMTKNQVS LYCLVKGFYP SD IAVEWE SNGQPENNYKT TPPVLDS DGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
105121 SEQ ID NO: 34:
AT GAGACC T GC GGACCTGCCCCCGCGCCCCAT GGAAGAA_T CCCCGGCGT CCAGCT C T GCCCCGA
CAGAGACGGAGGAGCCGGGG T CCAGT GCAGAGG T CAT GGAAGAAG T GACAACAT GC TCCTT CAA
CAGCCC TC T GT TCCGGCAGGAAGAT GACAGAGGGAT TACC TACCGGAT CCCAGCCC T GC TCTAC
ATACCCCCCACCCACACCT T CCT GGCCT T T GCAGAGAAGCGT IC TACGAGGAGAGAT GAGGATG
CTCTCCACCTGGTGCTGAGGCGAGGGTTGAGGATTGGGCAGTTGGTACAGTGGGGGCCCCTGAA
GCCAC TGAT GGAAGGGACAC TACCGGGGCAT CGGACGAT GAACCGCT GT CC T GTAT GGGAGGAG
AAGAGIGGTTGTGIGTICCTGITCTTCATCTGIGTGCGGGGCCATGTCACAGAGCGTCAACAGA
T T GT GTCAGGCAGGAAT GC T GCCCGCC T T T GC T TCATCTACAGT CAGGAT GC TGGAT G T T
CAT G
GAGTGAGGTGAGGGACT T GAC TGAGGAGG T CAT TGGCTCAGAGCTGAAGCACTGGGCCACAT T T
GC T GI GGGCC CAGGTCAT GGCAT CCAGC T GCAGT CAGGGAGAC T GGT CAT CCCT GCGTATACC
T
AC TACATCCC T TCCTGGT TCT ITT GC T TCCAGCTACCATGTAAAACCAGGCCTCA_T TCTCTGA_T
GAT C TACAGT GAT GACC TAG GGGT CACAT GGCAC CAT GG TAGAC T CAT TAGGCC CAT GG T
TACA
G TAGAATCT GAACTGGCAGACGT CAC T GGGAGGCC T GGC GACCC TGT GC TATAT T GCAG T
GCCC
G GACAC CAAACAGG T GC C GG GCAGAGGC G C T CAGCAC T GAC CAT GGT GAAGGCT T T
CAGAGAC T
GGCCC TGAGT C GACAGC TCTG TGAGCCCCCACAT GGT T GCCAAGGGAGT GT GGTAAGT T TCCGG
CCCCT GGAGAT C C CACATAG G T GC CAGGAC IC TAGCAGCAAAGATGCAC CCACCAT T CAGCAGA
GC T C T CCAGGCAGT TCAC T GAGGC TGGAGGAGGAAGC T GGAACACCGT CAGAAT CAT GGC TC T
T
G TAC T CACAC C CAACCAGTAGGAAACAGAGGGT T GAC C TAGG TAT C TAT C T CAA.0 CAGAC
CC C C
TTGGAGGCTGCCTGCTGGTCCCGCCCCTGG.ATCTTGCA.CTGTGGGCCCTGTGGCTA.CTCTGATC
TGGCTGCTCTGG.AGGA.GGAGGGCT TGT T T GGGT GT T TGT T T GAATGT
GGG.ACCAA.GCA_AGAGT G
144
CA 03173557 2022- 9- 27
WO 2022/150521
PCT/US2022/011504
T GAGCAGAT T GCCT TCCGCC T Gil TACACACCGGGAGAT CCT GAGTCAC CT GCAGGGGGACT GC
AC CAGCCCTGGTAGGAACCCAAGCCAAT T CAAAAGCAAT
105131 SEQ ID NO: 35:
ATGATGAGCTCTGCAGCCT TCCCAAGGTGGCTGAGCATGGGGGTCCCTCGTACCCCTTCACGGA
CAGT GC IC T T C GAGCGGGAGAGGACGGGCCT GACC TACC GCGT GCCCT C GCT GCT CCCC
GTGCC
C CCCGGGCCCACCCTGC T GGCCT T T GT GGAGCAGCGGCT CAGCCCTGAC GAC TCCCAC GCCCAC
CGCCTGGTGCTGAGGAGGGGCACGCTGGCCGGGGGCTCCGTGCGGTGGGGTGCCCTGCACGTGC
TGGGGACAGCAGCCCTGGCGGAGCACCGGTCCATGAACCCCTGCCCTGTGCACGATGCTGGCAC
GGGCACCGTCT TCCTCTTCT TCATCGCGGTGCTGGGCCACACGCCTGAGGCCGTGCAGATCGCC
ACGGGAAGGAACGCCGCGCGCCICTGCTGIGTGGCCAGCCGTGACGCCGGCCTCTCGTGGGGCA
GCGCCCGGGAC CT CACCGAGGAGGCCAT C GGT GGT GCCG T GCAGGAC T GGGCCACAT T C GCT GI
GGGTCCCGGCCACGGTGTGCAGCTGCCCTCAGGCCGCCTGCTGGTACCCGCCTACACCTACCGC
GTGGACCGCCGAGAGTGTT T TGGCAAGATCTGCCGGACCAGCCCTCAC T CCT TCGCCT TCTACA
GCGATGACCACGGCCGCACCTGGCGCTGTGGAGGCCTCGTGCCCAACCTGCGCTCAGGCGAGTG
C CAGC TGGCAGCGGIGGACGG TGGGCAGGCCGGCAGC T T CCTC TACT GCAAT GCCCGGAGCCCA
C TGGGCAGCCGTGTGCAGGCGCTCAGCAC TGACGAGGGCACCTCCTTCC TGCCCGCAGAGCGCG
T GGCT TCCCT GCCCGAGACT GCCTGGGGC TGCCAGGGCAGCATCGTGGGCT TCCCAGCCCCCGC
C CCCAACAGGC CACGGGAT GACAGT T GGT CAGT GGGCCC CGGGAGTCCCCT CCAGCCT C CAC IC
C TCGGTCCTGGAGTCCACGAACCCCCAGAGGAGGCTGCT GTAGACCCCCGTGGAGGCCAGGTGC
CTGGTGGGCCCTTCAGCCGTCTGCAGCCTCGGGGGGATGGCCCCAGGCAGCCTGGCCCCAGGCC
T GGGGTCAGT GGGGAT GI GGGGT CCT GGACCCT GGCACT CCCCATGCCC T T T GCT GCCC CGCCC
CAGAGCCCCACGTGGCTGCT GTACTCCCACCCAGTGGGGCGCAGGGCT CGGCTACACAT GGGTA
TCCGCCTGAGCCAGTCCCCGCTGGACCCGCGCAGCTGGACAGAGCCCTGGGTGATCTACGAGGG
CCCCAGCGGCTACTCCGACCTGGCGTCCATCGGGCCGGCCCCTGAGGGGGGCCTGGTT T TTGCC
TGCCTGTACGAGAGCGGGGCCAGGACCTCCTATGATGAGATTTCCITT TGTACATTCTCCCTGC
GTGAGGTCCTGGAGAACGTGCCCGCCAGCCCCAAACCGCCCAACCTTGGGGACAAGCCTCGGGG
GTGCTGCTGGCCCTCC
105141 SEQ NO: 36:
MR FKNVKK TALMLAMFGMAT S SNAAL FDYNATGDTE FDS PAKQGWMQDNTNNGS GVL TNADGMP
AWLVQG I GGRAQWTYS L S TNQHAQASS FGWRMTTEMKVLSGGMI TNYYANGTQRVLP I I SLDSS
GNIVVE.FEGOT GR TVT TGT AATF.YHKFET,VFT ,PGSNPS AS EYE-DGIKT, T RDNT OP T AS
KONTMTV
WGNGSSNTDGVAAYRDIKFE I QGDVI FRGPDRI PS IVAS SVTPGVVTAFAEKRVGGGDPGALSN
TND I I TRT S RDGC I TWDTE LNLTEQINVS DE FDFSDPRP I YDP S SNTVLVSYARWPTDAAQNGD
R IKPWMPNG I FYSVYDVAS GNWQAP I DVTDQVKERS FQIAGWGGSELYRRNTSLNSQQDWQSNA
K IRIVDGAANQ I QVADGSRKYVVTLS I DE S GGLVANLNGVSAP I ILQSEHAKVHS FHDYELQYS
ALNHT T TL FVDGQQ I T TWAGEVS QENN I Q FGNADAQ I DGRLHVQK IVL T QQGHNLVE
FDAFYLA
QQT PEVEKDLEKLGWTKIKT GNTMSLYGNASVNPGPGHG I TLTRQQNI S GS QNGRL I YPAIVLD
RFFLNVMS I YS DDGGSNWQT GS TLP I PFRWKSSS I LETLE PSEADMVE LQNGDLLL TARLDFNQ
IVNGVNYS PRQQFLSKDGG I TWSLLEANNANVFSNI STGTVDAS I TRFE QS DGSHFLL FTNPQG
NPAGTNGRQNLGLWFS FDEGVTWKGP I QLVNGASAYSDI YQLDSENAIVIVE TDNSNMR ILRMP
TLLKQKLTLSQN
105151 SEQ ID NO: 37:
TTGT CAAT CAAGATGAC T T CACAAC GAAGAAGAG CAT CGA T T CACAAGGAAACAGA T T C
TAATA
TAAAGGGAGTAGATATGCGT T TCAAAAACGTAAAGAAAACCGCT T TAAT GCT TGCAAT GT TC GG
TAT GGC GACAAGCTCAAACGCCGCACT T T TTGACTATAACGCAACGGGTGACACTGAGT TTGAC
AG T C CAGC CAAACAGGGAT G GAT GCAAGACAACACGAATAAT GGCAGC G GC GTTT TAACCAAT G
145
CA 03173557 2022- 9- 27
WO 2022/150521
PCT/US2022/011504
CAGATGGAATGCCCGCT TGGT TGGTGCAAGGTAT TGGAGGGAGAGCTCAATGGACATAT T CT C T
CTCTACTAATCAACATGCCCAAGCATCAAGT T TCGGTTGGCGAATGACGACAGAAATGAAAGTG
C T CAGTGGT GGAAT GAT CACAAAC TAC TAC GCCAAC GGCAC T CAGCGT GTCT TACCCAT CAT T
T
CA_T TAGATAGCAGIGGTAA_CT TAGT T GT TGAGTT TGAA_GGGCAAACTGGACGCACCGT T T TGGC
AACCGGCACAGCAGCAACGGAATATCATAAAT T TGAAT TGGTAT TCCT T CC T GGAAG TAACC CA
TCCGCTAGCT T T TACT T CGAT GGCAAAC T CAT TCGTGACAACATCCAGCCGACTGCATCAAAAC
AAAA T ATGA T C GTATGGGGGAAT GGC T CAT CAAAT ACGGA T GGT GTCGC CGC T TAT CG T
GATA T
TAAGT TTGAAAT T CAAGGCGACGT CAT C T TCAGAGGCCCAGACCGTATACCGTCCAT TGTAGCA
AGTAGCGTAACACCAGGGGTGGTAACCGCAT T TGCAGAGAAACGTGTGGGGGGAGGAGATCCCG
G T GC T C TGAG TAATAC CAAT GACATAAT CAC T CGTACCT CAC GAGAT GGCGGTATAAC T
TGGGA
TACCGAGC T CAACCTCAC T GAGCAAAT CAT GT CAGTGAT GAGT T TGAT T IC TCCGAT CC TCGG
CCTATCTATGATCCTICCTCCAATACGGITCTIGICTCTTATGCTCGATGGCCGACCGATGCCG
C T CAAAACGGAGATCGAATAAAAC CAT GGAT GCCAAACGG TAT TITT TACAGCGTC TAT GAT GT
T GCAT CAGGGAAC TGGCAAGCGCC TAT CGAT GT TACCGATCAGGTGAA_AGAACGCAGT T TCCAA
AT CGC TGGT TGGGGTGGT TCAGAGCTGTATCGCCGAAATACCAGCCTAAATAGCCAGCAAGACT
GGCAAT CAAAC GC TAAGAT C CGAAT T GT T GAT GGT GCAGCGAAC CAGATACAAGT T GC C GAT
GG
TAGCCGAAAATAT GT TGT CACACT GAG TAT T GAT GAAT CAGGT GGTC TAGT CGC TAAT C
TAAAC
GGT GT TAGT GC T CCGAT TAT CCT GCAAT C T GAACACGCAAAGGTACAC TCTT TCCATGACTACG
AACTICAATAT TCGGCGT TAAACCACACCACAACGT TAT TCGTGGATGGTCAGCAAATCACAAC
T T GGGC TGGC GAAG TAT CGCAGGAGAACAACAT TCAGT T TGGTAATGCGGATGCCCAAAT TGAC
GGCAGACT GCAT GTGCAAAAAAT T GT TCTCACACAGCAA_GGCCATAACCTCGTGGAGT T T GAT G
CTTTC TAT T TAG CACAG CAAACC C C T GAAGTAGAGAAAGACC T T GAAAAGC T TGGT T G
GACAAA
AAT TAAAACGGGCAACAC CAT GAGT T T GTAT GGAAAT GC CAGT GTCAAC CCAGGACCGGGTCAT
GGCATCACCCT TACTCGACAACAAAATAT CAGT GGCAGC CAAAACGGC C GC T TGATCTACCCAG
CGATTGTGCTTGATCGTTTCTTCTTGAACGTCATGTCTATTTACAGTGATGATGGCGGTTCAAA
C T GGCAAACC GGT TCAACAC T CCC TAT CC CC T T T CGC T GGAAGAGT T C GAGTAT CC
TAGAAAC T
C T CGAACC TAG T GAAGC T GATAT GGT T GAAC TCCAAAAC GGT GATCTAC T CC T TAC T
GCACGCC
T T GAT T T TAAC CAAAT CGT TAAT GGT GT GAAC TATAGCC CACGCCAGCAAT TITT GAG
TAAAGA
T GGT GGAA T CACGTGGAGCC TACT TGAGGC TAACA_AC GC TA_ACGICT T TAGCAA TA T C AG
TAC T
GGTACCGT TGATGCTTCTAT TACTCGGT TCGAGCAAAGT GACGGTAGCCAT =CT TAC T CTITA
CTAA_CCCACAAGGAAA_CCCTGCGGGGACAAA_TGGCAGGCAAAA_TCTAGGCT TAT GGT T TAGCT T
C GAT GAAGGGG T GACAT GGAAAGGAC CAAT TCAACT TGT TAATGGTGCATCGGCATAT TCTGAT
AT T TAT CAAT T GGAT T CGGAAAAT GC GAT T =AT T GT TGAAACGGATAAT TCAAATATGCGAA
TTCTTCGTAT GCC TAT CACAT T GC TAA_AACAGAAGC TGACC T TAT CGCAAA_AC TAA
105161 SEQ ID NO: 38:
MVGADP TRPRG PL SYWAGRRGQGLAAI FL LLV SAAE SEARAE DD FS LVQ PLVTME QLLWVS GKQ
I GSVDT FRI PL I TAT PRGT L LAFAEARKKSAS DE GAKFIAMRRS TDQGS TWS S TAF I
VDDGEAS
DGLNLGAVVNDVDTGIVFL I YTLCAHKVNCQVAS TMLVW SKDDG I SWS P PRNLSVD I GTEMFAP
GPGS G I QKQRE PGKGRL IVCGHGTLERDGVFCLLSDDHGASWHYGTGVS G I PFGQPKHDFIDFNP
DECQPYELPDGSVI INARNQNNYHCRCRIVLRSYDACDTLRPRDVTFDPELVDPVVAAGALATS
S G IVFFSNPAHPE FRVNLTLRWS FSNGT SWLKERVQVWP GP S GYS SL TALENS T DGKKQ PQL F
VLYEKGLNRYTES ISMVKI SVYGTL
105171 SEQ ID NO: 39:
MTVQP S PW FS DLRPMAT C PVLQKE TL FRT GVHAYR I PAL LYLKKQKT L LAFAEKRAS KT
DEHAE
L IVLRRGS YNEATNRVKWQPEEVVTQAQLE GHRSMNPCP LYDKQTKT L FL FFIAVPGRVSEHHQ
LHTKVNVIRLCCVSSTDHC-IRTWSPIQDLTETT IC-1S THQEWAT FAVGPGHCLQLRNPAGS LLVPA
YAYRKLHPAQKP T P FAFC F I S LDHGHTWKLGNFVAENS LE CQVAEVGT GAQRMVYLNARS FLGA
RVQAQSPNDGLDFQDNRVVSKLVEPPHGCHGSVVAFHNP I SKPHALDTWLLYTHP T DS RNRTNL
146
CA 03173557 2022- 9- 27
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PCT/US2022/011504
GVYLNQMPLDPTAWSEPTLLAMGICAYSDLQNMGQGPDGS PQFGCLYE S GNYEE I I FL I FTLKQ
AFPTVFDAQ
105181 SEQ ID NO: 40:
MEEVPPYSLS S TLFQQEEQSGVTYRI PAL LYL PPIHT FLAFAEKRTSVRDEDAACLVLRRGLMK
GRSVQWGPQRL LMEAT L PGHRTMNPC PVWEKNT GRVYL F F CVRGHVT E RCQ IVWGKNAARLC F
L CSEDAGCSWGEVKDL TEEVI GSEVKRWAT FAVGPGHGI QLHS GRL I I PAYAYYVSRW FLCFAC
SVKPHSLMI YS DDFGVTWHHGKFIEPQVT GECQVAEVAGTAGNPVLYC SART PSRFRAEAFS TD
SGGCFQKPTLNPQLHEPRTGCQGSVVS FRPLKMPNTYQDS I GKGAPAT QKCPLLDS PLEVEKGA
E TPSATWLLYSHPTSKRKRINLGIYYNRNPLEVNCWSRPW I LNRGPS GYS DLAVVEEQDLVACL
FECGEKNEYER I DFCL FS DHEVLS CEDC T S PS S D
105191 SEQ ID NO: 41:
ME TAGAP FC FHVDS LVPC S YWKVMGP TRVPRRTVL FQRE RT GL TYRVPALLCVPPRP T L LAFAE
QRL S PDDSHAHRLVLRRGT L TRGSVRWGITSVLE TAVLEEHRSMNPCPVLDEHS GT I FL FFIAV
L GHT PEAVQ LAT GKNAARLC CVT S CDAGL TWGSVRDL TE EAI GAALQDWAT FAVGPGHGVQLRS
GRLLVPAYTYHVDRREC FGK I CWT S PHS LAFYS DDHG I SWHCGGLVPNLRSGECQLAAVDGDFL
YCNARS PLGNRVQALSADE GT S FL PGELVP TLAE TARGCQGS TVGFLAP PS IEPQDDRW T GS PR
NT PHS PCFNLRVQES S GEGARGLLERWMPRL PLCYPQSRS PENHGLE P GS DGDKT SWT PECPMS
S DSMLQS P TWL LYSHPAGRRARLHMG I YL SRS PLDPHSW TE PWVIYEGP S GYSDLAFL GPMPGA
SLVFACLFESGTRTSYEDI S FCLFSLADVLENVPIGLEMLSLRDKAQGHCWPS
105201 SEQ ID NO: 42:
GGGTCACAT GC TGATGGAC TAATTGGAGTCGCGGCAGCGCGGGCTGCGGCCCCCAAGGGGAGGG
GTCGGAGTGACGTGCGCGCT T TTAAAGGGCCGAGGTCAGCTGACGGCT TGCCACCGGTGACCAG
T TCC T GGACAGGGATCGCCGGGAGC TAT GGTGGGGGCAGACCCGACCAGACCCCGGGGACCGC T
GAGC TAT T GGGCGGGCCGT CGGGGTCAGGGGC TCGCAGCGATC T TCC T GC TCCTGGT GT CCGCG
GCGGAATCCGAGGCCAGGGCAGAGGAT GAC T TCAGCC T GGT GCAGCCGC T GGTGACCAT GGAGC
AGC T GC TGT GGGT GAGCGGGAAGCAGATCGGC TCIGTAGACAC T TTCCGCATCCCGC T CATCAC
A_GCCACCCC T C GGGGCACGC T CCT GGCC T I CGC T GAGGC CAGGAAAAAATC T GCAT CC
GATGAG
GGGGCCAAGT TCATCGCCATGAGGAGGTCCACGGACCAGGGTAGCACGTGGTCCTCTACAGCCT
T CATCGTAGACGATGGGGAGGCCTCCGAT GGCC T GAACC T GGGCGCT GT GGT GAACGAT GTAGA
CACAGGGATAG T GT TCC T TAT CTATACCC TC T GT GC TCACAAGGTCAAC T GCCAGGT GGCCTC
T
ACCAT CT I GG T T I GGAGTAAGGACCACGCCAT T I CC TGGAGCCCACCCC GGAATC TC T C T
GT GG
ATAT T GGCACAGAGAT GT T T GCCCCT GGACC T GGC TCAGGCAT TCAGAAACAGCGGGAGCCT GG
GAAGGGCCGGC TCAT T GT GT GTGGACACGGGACGC T GGAGCGAGATGGGGTC TIC T GT C TCC TC
AGT GATGACCACGGTGCC T CC TGGCAC TACGGCAC T GGAGT GAGCGGCAT TCCCT T T GGCCAGC
CCAAACACGAT CACGAT T T CAACCCCGACGAGT GCCAGCCC TACGAGC T TCCAGATGGCTCGGT
CATCATCAACGCCCGGAACCAGAATAACTACCATTGCCGCTGCAGGATCGTCCTCCGCAGCTAT
GACGCCTGT GACACCC TCAGGCCCCGGGAT GT GACCITCGACCC TGAGC TCGTGGACCC T GT GG
TAGC T GCAGGAGCACTAGCCACCAGC I CC GGCAT I GICT TC T IC TCCAAT CGAGCCCAC CCT GA
GT TCCGAGT GAACCTGACCC T GCGC T GGAGT TTCAGCAAT GGTACATCC T GGCAGAAGGAGAGG
GTCCAGGT GT GGCCGGGACCCAGCGGC TAC TCGTCCC T GACAGCCCT GGAAAACAGCACGGAT G
GAAAGAAGCAGCCCCC GCAGC TGT TCGT TC T GTAC GAGAAAGGCCTGAACCGGTACACCGAGAG
CATC T CCAT GG T CAAAAT CAGCGT CTACGGCACGC ICI G_AGCCCCGT GC CCAAAGGAC ACCAAG
T CC T GGTCGC T GACT T CACAGCTC TC T GGACCATC T GCAGAGGGTGCC T GAAACACAGC T CT
IC
C TC T GAAC TC T GACCT T T T GCAAC T TC TCATCAACAGGGAAGTC TCT T CGT TAT GAC T
TAACA_C
CCAGCTTCCTCTCGGGGCAGGAAGTCCCTCCGTCACCAAGAGCACTTT TT TCCAGTAT GC TGGG
GAT GGCCCC T GTCCAT TC T C T TCCAGGACAACGGAGC T GT GCC T TIC T GGGACAGGAT
GGGGGA
147
CA 03173557 2022- 9- 27
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GGGGC TCCCCC T GGAGAGAT GAACAGATAC GAAC TCAGG GAAC T GAGAAGGCCCGGT GT CCTAG
GGTACAAAGGCAGGTAC TAGATGT GAT T GC T GAAAGTCCCCAGGGCAGAGT GTCC T T T CAGAGC
AAGGAT.AAGCACACCTA.CGT GTGCA.CC T T T GAT TA.T T TAT GAATCGAAATAT T T GTAA.0 T
TAAA.
AT TIT TGATGCAGAAAAA_GCGTTIGTGGAGICTGIGGITC TGTCTGCT CAGGCCT TCCCAAT TG
CC TCC T GGAGAGACAG GAAG GCAGC T GGAAGAGGAGCCGAT GTACT TAC T GGGAAGCAGAAACC
CC TAGAT TCCATCCTGGC T GC TGC TGT T T GCAAGT GTCAAAGAT GGGGGGGCGT GT T TATAT T
T
T_ATAT T TC T AAGAT GGGGT GGCAT AGGAAATAGGGAACAGAT GT GTAAAAC CAGAT GGGAAGGA
CAGTC TGT GAGAAAGGA.GCAAGCAGT T GC T GCAGGTGT GGGAGAGCAAAGGCCTIC TCCACGT G
G.AA_AG.AGCCCAGATGGACGC T.AAGC.AT GT TGGGC.ACCIGTAACCCCGCACTCGCTGGA.CTGACG
GT GTAGCTCAGT GGTGGAGC TAGTAC T T GGAACGCC TAAGAC TC TGGGT TCAGTCCTTGGGGGG
GGGGGTAT GT GT T TAT T GAGAGGAAGGT G TACGTAC TGTAGGT CAGAGGACAGCT TAC T GGAGT
TGTCTCTCTCCTTC.ACGCTGTGAGTCCTGIGGAATGACCTCAGGIGTCAGAGTTGGGGGCAGGT
GCCTTTGCCAGCTGAGCCATCTTGCTGTCTCTGCTTTATTTAAAGAT
A_TTAAGGICTGAGGGATTCGGGCTGCGTICATTICAATTAGAGGGICA_TATTICTTTTGACATT
T C T TC TCTAA.GAAATGT TAAGATC.AT T T GT TC T GTGTGATAGAGGTATAGC TCCAT T
GTATGTC
AGCAGTGAGGGAT CCT GT GCAT T T TAT CCAGAGT T T GTACGGT GT IC TAGGGGCT GC
TAGTGCA
GCCCAGTGCTAAACACTTCAGCATGCACAAGGCCTCAATCAGTGCATGCATGTGCACACACACA.
CAGACACACACGTACACACT GACACAGG TACACAAATACACAC T GGC C CACAT GTACACAT C GA
CT CACAG G TACACAGAC C CAC ITT GACACACATATACACAGACACAAAC G CAC T GGCACACACA
T A T AC.ACAG G CACACA T G GA T AGA.T GGACA.CA.CGT
GTACACAT.ACA.CA.CAC.ACACAG.AAATACA
AATGTTCAGGTTTTCT
T TAGAGACGT GT T GACT TCAT T T T TAGCAAAAAT C
C T GT CATGTAT CT TAAAGT GGAT T GAACC CAC TAT GTAGCCCAGGCT GGCC T CCAAAT
GGGCAT
CC T TC TGGC T CAGTGICCCGAGGGCTAGGATAACAGGAGTAT GCCAT GACACCT GCC TAATAGA.
AA TTTICAAAATTGITTGT T TGAAGGTGACTCTTACTATATTGCCTAACTGATCTCCAGTTCGT
GAAAT CC T CC T GC C T CAGAAC CAG GAC T G T CAATATAAC C CAC CAAGACAGGCCAACAT
TCACA.
A.T T GAT TGT TAGT T TGTGGT C TG.AATCAAGGIC T TATAC T GTAGCCCA.GGC
TAGCCCGGAATA.0
ACGAT.ATC TCCAGTGC T TCAGATCCTCAGT TC TAAC TAAGCAT GGCCACATCCAT GT T TAACTG
CAAATTTGATGTTACCATGGTTTGGTTTGGTTTGGTTTGGTTTGGTTTGGTTTGGTTTGGTTTT
T TGGCCATTT T T T TT T TC T C_ATGC T GAGGCC =GT GC TC TCAAGTIGGGGAGACAGCA_T
GGAGG
GTAGCTGCAA.CTGTAACCCC.AGTTCCAGGGGACCTGA.CACCCTCTGGCCTCCACAAGTATTAGG
CAGAT C TGT GG T GCACAGAC_ATACAAT CAGGCAAAA_TAT T CATACACATAAAATAAAA_TAAT T T
AAAACAAAAGCAAAAAT CAGGACC TAAGAAAAAAAT C TAT T CC T GAT TCTTT TAT GT T T T GT
T T
GTAT T TTATCAAGACAGGGT TGITTCTCTGTATAGCCCTGGCTGICTTGGAATTCACTCTGTAG
A.CCAGGCT GGCC TCAAAC T C.AG.AAATCC TCC T GCCT T T GCC T TCC.AAGT GC T GGAAT
TAAAGGC
AT GCGCCACC
105211 SEQ ID NO: 43:
GACATGACCCAAACGGCCCCTGGCTGCAAGGTAATATCGGAAGTTGACTAAGAATGGACGCCCC
A.CCACTGACTGACCCGCCCCCTGA.GTCTG.AG.ATTGG.ACT T GTC TCTGGATACAGTCATAC TT T G
A.GGTACTACAAGTTAGAAA.CTGITAGGTTACICAGTTCAGICC.ATGACAGTCCAACCT T C TC CA
TGGTTTTCCGATCTCAGGCCCATGGCGACCTGCCCTGTCCTGCAGAAGGAGACACTGTTCCGCA
C.AGGCGTCCA.T GC T TACAGAATCCCT GC TC T GC TC TA.CC T GAAG.AAGCAGAAGACCC T GC
TGGC
CTTTGCGGAAAAGCGAGCCAGCAAGACGGATGAGCACGCAGAGTTGAT T GTCCT GA GAAGAGGA
AGC TACAAC GAAGCCAC CAA.CCGT GTCAAGT GGC.AGCCT GAGGAAGT GGT GACCCAAGCCCAGC
T GGAAGGGCAC CGCTCCAT GAAT CCAT GT CCC T T GTAT GACAAGCAAACAAAGACCC T C T TCC
T
T TTCTTCATCGCTGTCCCTGGGCGTGTATCAGAACATCATCAGCTCCACACTAAGGTTAATGTC
ACACGGCT GT GC T GTGICAGCAGCAC T GACCAT GGGAGGACC T GGAGCCCCATCCAGGACCTCA
CAGAG.ACCA.CCAT TGGCAGCACTCA.TCAGGAAT GGGCCACAT T T GCT GT GGGTCC T GGGCAT T G
T C T GC.AGC T GCGGAACCCAGC TGGGAGCC T GC T GGTACC T GC T TATGCC TACCGGAAAC T
GCAC
CC T GC TCAGAAGCCTACCCC C ITT GCC T TC T GC T T CAT CAGCC T TGAC CAT
GGGCACACATGGA
AAC TAGGCAA_C T T TGTGGC T GAAAAC TCAC T GGAGT GCCAGGT GGCT GA GGT TGGCAC T
GGAGC
148
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T CAGAGGAT GG TATAT CT CAATGC TAGGAGC T T CC T GGGAGCCAGGGT C CAGGCACAAAGTCC T
AAT GATGGTC T GGAT T TCCAGGACAACCGGGTAGT GAGTAAGCT TGTAGAGCCCCCCCACGGGT
GTCAT GGAAGT GT GGT TGCC T TCCACAACCCCATCTCTAAGCCACAT GCCT TAGACACAT GGCT
TCTT TATACACAC CC TACAG_AC T CCAGGAA_TAGAA_CCAA_CC T GGG TGTG TACCTAAA_C
C_AGAT G
CCACTAGATCCCACAGCCT GGTCAGAGCCCACCCT GCT GGCCAT GGGCATCT GT GCCTACTCAG
ACT TACAGAACAT GGGGCAACGCCCT GAT GGCTCCCCACAGT T T GGGT GTCT GTAT GAATCAGG
T_AACT AT GAA_GAGAT CAT T T T CC T CATAT T CAC C C T GAA_G CAAGC TTTCCC CAC T
G TA_T T T GA T
GCCCAGTGATCTCAGTGCACGTGGCCCAAAGGGCTICCT T GI GCT TCAAAACACCCAT C T CTCT
T T GCT TCCAGCAT CCTCT GGACTCT T GAG T CCAGCTCT T GGGTAACT T CCT CAGGAGGAT
GCAG
AGAATTTGGTCTCTTGACTCTCTGCAGGCCTTATTGTTTCAGCCTCTGGTTCTCTTTTCAGCCC
AGAAATCAAAGGAGCCIGGCTITCCTCAGCCIGTTGGCAGGGCAGGTGGGGACAGTATATATAG
AGGCTGCCATTCTGCAIGTCGGITGTCACTATGCTAGTTTAACCTGCCTGTTICCCCATGCCTA
GTGTTT GAAT GAG TAT TAATAAAATAT C CAAC C CAG C C CA TTTCTTCCTG GAAAAAAA
105221 SEQ ID NO: 44:
ACT GCGCGGT GAAGGGGCGT GGCCTGGCCGGGGAGGT T GACACCCAGACGCT GCTCTCAGTCCT
C T GGCGCCT GC TCCCCAGCGCAT TCCT TC T GCTCCT GGGATAT T TGTC T CAT TACT GCCAGT
TC
TTGCGCAGCGGTCACTGGGTTCGITTCAGCGICTGIGGITTCTGICGCTGTTATCCAGTCTCCA
TCGCCCCAGCTCAGCTICAGGCCTICTTCCGAGACTCCACGGGAGAGCCCAGAGAGCCTCCGGA
GCCGAAGC CAT GGAGGAAGT CCCACCCTACTCCCTCAGCAGCACCCT GT TCCAGCAGGAAGAAC
AGAGTGGGGTGACCTACCGGATCCCAGCCCTGCTGTACCTTCCTCCCACCCACACCTTCCTGGC
CTTT GCAGAGAAGCGGACCT CAGTCAGAGAT GAGGATGC T GCCT GCCT GGT GCTCAGACGAGGG
C T GAT GAAGGGGCGCTCT GTACAGTGGGGCCCCCAACGGC TACT GAT GGAGGCCACAT TACCTG
GGCATCGCACCATGAACCCCTGCCCTGTGIGGGAGAAAAATACTGGCCGTGTGTACCTGTTITT
CATCT GIGT GCGGGGCCAT GT TACTGAGAGGTGCCAGAT T GT GT GGGGCAA_A_AAT GCCGCCCGT
CTCTGCTICCTTTGCAGTGAAGATGCCGGCTGCTCTIGGGGTGAAGTGAA_A_GACTTGACCGAGG
AGGTCAT T GGC TCAGAGGT GAAGCGCT GGGCCACAT T T GC T GT GGGCCCAGGTCAT GGCATCCA
GCTACACTCGGGAAGGCT GATCATCCCCGCCTATCCCTAC TAT GICTCACGT TGGT T T C TCT GC
T T T GCGTGT T CAGTCAAGCCCCAT TCCCT GAT GATCTACAGT GATGAC T TTGGAGTCACATGGC
ACCATGGCAACTTCATTGAGCCCCAGGTGACAGGGGAGTGCCAAGTGGCCGAAGTGGCTGGGAC
GGCT GGTAACCCT GTGCTCAC TGCAGT GCCCGAACACCAAGCCGAT T T CGAGCAGAGGC T TT TA
G TAC T GATAG T GGTGGC T GC T T T CAGAAGCCAACCC TGAACCCACAAC T C CAT GAGC C T
CGAAC
CGGCTGCCAA_GGTAGIGTAGTGAGCTICCGGCCTITGAA_GATGCCAAA_TACCTATCAA_GACTCA
AT T GGCAAAGGT GCTCCCGC TACTCAGAAGT GCCCICT GC T GGACAGT CCTCTGGAGGT GGAGA
AAGGAGCT GAAACAC CAT CAC CAACAT GGCTCTTGTACT CACAT CCAAC TAG CAAGAG GAAGAG
GAT TAACCTA_GGCATCT ACT_ACAACCGGAACCCCT TGGAGGT GAACT GC T GGTCCCGCCCGT GG
ATCT T GAACCGT GGGCCCAGT GGCTACTC T GATCT GGCT GT T GT GGAAGAACAGGACT T GGT GG
CGTGTTTGTTTGAGTGTGGGGAGAAGAATGAGTATGAGCGGATTGACTTCTGTCTGTTTTCAGA
C CAT GAGGTCCT GAGC T GT GAAGACT GTACCAGCCC TAG TAGC GAC TAAAGC CAAAT CAAGACG
GAT GAG T GAG G C C CAGC TTCCCACAGAAAGGAAT GGCAGC TACAGCCAG GG TAACAGAG CTCTC
TGATGICTAGAGAAAACTCTAAAAACTAATAATCTGCTCCTTGAATTTTTTCACTTTTCCCTIC
AAT GAG CAT GG T GAAAAT T G T GC CATAT C T IAEA TAAC GAG GCTCTT GAAC T GG GAG
T T T GAAT
CTCTTCTCTTCCCATTAAAAGGAGAGGCCATGTGCTCGCT TCGCGTTCGACAAAGCCTGGATTC
TGATCTTGAGTGGAAGCCACAGGCTTGTCTITTCCAATGGTTCACTGCTCACCTGAGTATTAGG
TGATGIGTAGGTGCCTIGGCCAGAAGAAAGATCTGIGTTGTTGTATTTTTTTAAATTTATTTAT
T TACTATATGTAAGTACACTGCAGCTGTCTTCAGACACACCAGAAGAGGGCGTCAGATCTCATT
AGAGATGGT T G T GAGCCACCATGT GGT T GC T GGGAT T T GAAC T CAGGAC C T T
CAGAAGAGCAGT
CAGT GCTCT TAAC TACT GAGCCATCTCTCAAGCCCCGCAT TGCTGTAT T TTTAATAAGAAAAAT
GCCCTTATCCT TCCAATAATGCCTGGAGCTGTACAAATTCTCTGTCTTAGAAGACTTGAGAAAG
CAGAACTGTAAGGICAGATGCTITCTCCAGCCITGATGCTGTGTTCCACCTTCCCTTCCTCATC
CAGAAAACAGTTACTAGGGAGAAAATGAGAAACCCATGCCAGCTGCCCTTGATGATGGTTGATA
149
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ACGGTGCT TAT TGCTT I TGATGTC.AT TACCTCTGT TAGAGATGAATCAGAGTCAGA.GGT CCT TA
GCTGCATCCACCCATTICCAGGGGGACATTCTAACACTGCTGAAC.AGTCAGCTAAAA.TGAGAGC
T GTGTGICCTAGCCTGA.T T CC.AGGITAGICATGATGCT T CCTGGAGCT GGGCTTT TAT C TAATC
C CAGGAGCCAT C TAGGGGAGGCT CAGAGC TAGCAGGTGATCT T CCTGAGAT GGT T T CAC CGT GA
CAGGT GAAC CAT GAGC CCTTC CAAG CAAG GC CAAAG GACAACAT TATAGGAAAGAT T T C TAG
TA
T TAATATGCCT TTICTCTGTGICTGTACTGICTIGTAGTGATGCTATATAGACAAATAGATGAT
TTCTTATTTTTTGTTTGTTTGTTTGTTTTTTTGTTTTTCTGTAGCCCTAGCTGTCCTGGAACTC
ACTTTGTAAACCAGGCTGGCCTCGA.TCTCAGAAATCCGCCTGCCTCTGCCTCCCGA.GTGCTGGG
AT TAA_AGGT G T GCACCACCA.C.ACC T T.AAT G.AT G.AT CC TAT.AAG TAT T C C TAAAAT
TATAC TAG T
AAT TAT TAAC T COTT TA.TAATAGG.ACTGC TAT TAAAGCCC TCGCTGATAT GAAAAC TACAGT GA
GAACTCTGC CAGTCT TCACAT GT C.ATAA.T TACT TCTGAGATAG.AAAGCAGGCAT T TACAACT TA
GAACACAT TTCT TAGAGC T G TAAAACAAT TAAC TAGAGG T CATAAAA.GGGAATGAAA.GAT T TAT
T GTA.GGIGCTAGG.ACA.GAA.CATAAAATA.T I GAG I GGGC T TAT C T.ATAT GAAAC T T CAT
T GTTAA.
CTTTT ACAC AAGAAT TA TGGT TT T TAACT T TCAGTGAACC TGC GGAGC TAGTGACA GAAGAGAA
ATGTCTAGTTAGATAA.CTACTCTT.AATGGAAATTCACATAAACATCTGT TGC CATCT T C T TT T T
GAAT T TAT G T T TAAACTTGT GAAT GT T TGAAT TAGACAC TAC GC GAG
CACATAGAAAATAAAGA
AC TAAGCG T GAA
105231 SEQ ID NO: 45:
GGACAGTGT GCAT CACGGAGC IT GTGGCC CAGAC I GTGCC T GGCAGAC C CAGAGGA.CC TAAGGC
T TGGCTCTAGTGGTGGTCAGCACAGCCCTCGGTGGTCTGCGGAGCCTGATATTGCTTTACGTAA
GGGCTGTTCTGCTGTGCATCTCCTGTGTCTGAAGCTATTCGCCATGGAGACTGCTGGAGCTCCC
T TCTGCTICCATGIGGA.CTCCCIGGTACCTTGCTCCTACTGGAAGGTTATGGGGCCCACGCGTG
T TCCCAGGAGAACGGTGCTCT TCC.AGAGGGAAAGGACGGGCCTGACCTACCGTGTGCCTGCGTT
ACTCTGIGTGCCTCCCA.GGCCTACTCTGCTGGCCITCGCGGAA.C.AGCG.ACTT.AGCCCTGATGA.0
TCCCATGCCCACCGCCIGGTGCTAEGGAGGGGCACGCTGACCAGGGGCTCAGTGCGGTGGGGCA
CTCTGAGTGTACTGGAGACTGCAGTACTGGAGGAGCACAGGTCTATGAACCCTTGCCCGGTGCT
GGAT GAGCAC T CT GGTACCAT CT T CCTCT TCT I CAT TGC CGT GC TGGGC
CACACACCGGAGGCC
GTGCAA_ATCGCC.ACTGGCAA.GAACGCTGC TCGCCTCTGC TGTGTGA.CC.AGCTGTGA.CGC TGGCC
TCACCIGGGGCAGIGTICGAGATCTCACTGAGGAAGCCAT TGGTGCTGCATTGCAGGACTGGGC
C.ACCTITGCTGTGGGICCGGGCCA.TGG.AGTTC.AGCTGCGCTCGGGICGCCTGCTIGTTCCTGCT
TACACC TAT CAT GTGGA.CCGACGGGAAT GT T T T GGCAAGATCT GCTGGACCAGT CCCCAC TCCT
TGGCATTCT.ACAGTGATGATCATGGGATCTCCIGGCATTGTGGAGGCCT TGTGCCCAA_CCTACG
CTCTGGAGAGTGCCAA.CTGGCTGCGGT.AGATGGAG.ACTT TCTCTACTGTAA.TGCTCGAAGCCCT
CTGGGTAACCGTGTGCA.GGCACTGA.GTGCTGA.TGAAGGCACGTCCITCCTACCAGGGG.AGCTGG
T GCC T ACAT T GGCAGAGACGGCT CGT GGT I GCCAGGGTAGCAT T GTGGGCT T CCTAGC T
CCACC
C TCAATCGAGCCTC.AGGAT GACCGGT GGACAGGGAG T CC TAGGAACACCCCACAT T CC C CAT GC
T TCAATCTGAGAGTACAGGAGICTICGGGGGAAGGIGCCAGAGGICTTCTTGAACGTTGGATGC
GCAGGITGCCTCTCTGGTACCCACAGTCCCGG.AGCCCAGAGAATC.ATGGCCT.AGAGCCTGGGIC
A.GATGGAGA.TAAGACATCCT GGACTCCGGAATGTCCTAT GTCCTCTGAT TCCATGCTTCAGAGC
CCCACATGGCTACTATATTCCCACCCAGCAGGGCGTAGAGCTCGGCTCCACATGGGAATCTACC
TGAGCCGATCCCCCTIGGATCCCCA.CA.GCTGGACA.GA.GCCCTGGGTGATCT.ATGA.GGGCCCCA.G
TGGCTACTCTGACCTTGCCTTTCTTGGGCCTATGCCTGGGGCATCCCTGGTTTTTGCCTGTCTG
T T TGAGAGCGGGACCAGGAC T TCCTATGAA.GACAT T TCT T TTTGCTTGT TCTCACTGGCGGATG
TCCTGGAGAATGTGCCCACTGGCTTAGAGATGCTAACTCTCAGGCATAAGGCTCAGGGGCATTG
CTGGCCCTCT TGATGGCCTCACCCTCTCGTAGCCGCCTGGAGAGGAAGGGTAGACTATATAGAG
GAGGTTAGGGGTAGGTCAGCATGATGCTAGGATGGAGAGAGCTCTGTCCCCTCGTGGATGGTGG
TGGTG.ACTGA.CCCGGGGGGCCAGCTGCTITCTGAGTGCA_AATG.AG.AAAAAT.AAAGA.GCTGCGCT
GTGACITTTCTTTCCACATCAAA.GCTIGGGIGICAGTGCTTTAGCTTGATGCTCTGATCACCAT
GCAAA.TCTTCCACCGGCGCCT TGCTCAGCTITCATATCCCAA.GGGIGCCTGGGAGGAA_GGCAA.0
AGGGACAGT GGACATCAC T GCACCAC I I T CCACGACCCT GT GT GCCAAC CT CAGCCAC T TTGAA
150
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A.CAT GC TGA.T GAC TGAGGT C T GIT CAC TTICT TAA.T T TCAA.GCAGGAGAA.GCAGGT T
GGGGAGC
CAGCCTCCCCAGCTAGAGGGGACAGAA.CTTGACTTGAGCAGGGGGGTACCTCCTAGGACCTGCT
CCATGTGCCTACTICTITACCCTICTCTAGAGAGGGCTCTTGTCCIGTCAG.AGCTGTTTTCTCC
CTTCTCTIGTTTTTICITTTTCAAGACTGITTCTCTGIGTTAGCCCTGGCTGICCTGGATCTCA
CTCTGTAGATCAGGCTGACCTTGAGTICAAA.GCTCCATCTGCCTCTACTTCTCACATTACTGIG
AT TAAACCCATATACTACCAC TGCCT GGT GCCC T T T TGTAT T TCT TAT TAAA.CT CC TAA.T
CTC T
GAT T ATAAAA_ACAGTC T GT G T GGGC T GGAGT GAT GGC T TAC T CAGTAAA GCACT T GCC
AT GGAA
T C T GGGCAA.T C T GAGT T T CAT T TT TAGCAT CCIGTAAAAA.T CCCAA.T T T GAT
GGTGTAC T TGTA
A.T GT C.AGCAT GGAGAGGCAGAGATA.GGTA_AGTTCCCC.AA.GAC T C TIT GA_AC C GACAGC T T
GGCC
T CAC T GGCACAT T CCAGGT C T CAGTGAGAGACCC T GC C T CAAAA.TACAAA.GAAAGAGC T GC
T GA
AGAGT GGGT CAGAGT T GACC T CT GAT C T CCGGAA.GTATAT GAT.ACACACCCGTGCAT GCACT C
T
TCCT TACAA_AA.TAAAAA.GCAAAA.CAAAA.0 C C CAAC.AGG TA TAT GGC CAT T T TAGAAAAA.T
TAGA.
AGATTTAGAAkGCTATACATAAATGACCTAAGAAAATCTT TAC TGTICTGGGCAC T
ATCCCTATCAAACCACTGTGTICITTGGCCAAGCCITGGGGTGGACACTGTTITGAGGTGGGIC
CTGTTATCTCCACTAGGTAGTGGAGTTTTGTGTCAGACTAA.CTGGGTCTTAA_AGCTGTCTTTAA.
GGCCATCAGGAGCTACTGACT TGCCTGCCTCAGCAGAGCATATCCTGAA.GGTCGGGGT TAAGTC
TCCTTCCCGAGCGAGTIGCCTICC.AGIGGGCCCCTGGACTCCTAGGTCCTCAGCGCTCATCAGC
T GCCAA.GGAC T C T GAGGGAA.T GICCT C T CAC T GIGGCCCCGAAA.GGTAGGGGAGGGGGAT GT
GC
T TAGGC T TAGGACAGGGT CC T &TT T CAGT C T GCC T TCAC T GT TAGTAGCAC T GT
GCCACATGGC
A.CAGAC TGGGCG.AGCT T TAA_AGGA_A.GGAGGT TGATAT T GG T T CCCA.0 TTCT GGGGA.T
CAT Gal T
GAGCAGCC T T G T C TGAT GAT GGTT GT C T T GAT GGTAGAT CGT GAGGTAG T T GAT
GAA.GG TAT GA
CATGGTGAGAAA.CTCTGTGTGIGIGTGT TAT TTICTCTGTGT TCTACCTATACATCTATCTATG
TATATATGTA.T C TATC TAT C TACC T GGAGGC TGGAGAGATAGC T TAGT GGT TAAGAA.CAT T T
GT
T GT T C T TGCATAGTCC T GGAT TTAAATTT TCAGCACCCACATGGCAGCTCACAACAACCCATAA
AT CCAGTT T CAGAGGAT CCA.ACCICT GATATAC CAT GTCAGCCAGAGCAGACACGGC T GAAGGT
GGTT T GAT CC C CG TAT GGAGAGGT GACAA.T T GGGAA.GAGAGAA_AGAT CAA.CT TAA.CCA.T
GCAA.G
GAA.CAGGAA.G T TAAAT AC T GAACAGGGAA.GGTAAA.GGCAGGAAGTAGA.T GTAGAGGGCA.AAT CA
AT GAAA.CCCAAA.CATACCCAAAT TAC GC TAAA.CACACAC T GACAT GC CAA.T TAAAA.G GA
CAAA.T
TGGCTCCACTGGCAAAACCAAAACAGACACTGAAGATCCAA_ACAGICACATGCCAACTACCGCG
GAGGG.AGACAGACACAGAGA.AGACCGT GACA.GA.CA.0 TIGGACAC TCTT GAG.AGT GGAT GT GCAG
GAAGAGAGC T C T GCCAGT GGAGAAGAAAG CAC T CAGAA_GAAAGT GACAGCAGCT GTAAAT TTGT
AT T C T GC TAA.T GT TAT GTTC CAAA.GT T GAAA.GCAAAA.T T G TACCAAT T
CATAAGAA.CAAA.CAGG
CTGACTCTCAGTTCTGACTCAACGTCTCTCAGTAACTGACGGGGCCACCAGGCCAAAGGAGAGT
CGGC T CAGAA.G GG T GCA.TAG C CAC GC CA_AA.T CA_AA.T.AA.G CAAG TACAA.0 C
GGCAGGC T C TAT IT
CTAGCACAAA.GGGGICIGTGCCTCA.TTCTGTGCTTGGGICAGAGCTTGGGTCTCTCATTTGGAT
G TAA.GTGGT G TAG T GGAGAA.G CAG GA_AA.T A_AT C C G G.AGC GCAT.AT T T T GAT T
T TAA.CA.TAAGT G
CTGATTIGGGAGGGAGITTTGICAAA.TTGIGTTITTACAA.TGTTITTTTTTTITTAAA.TGATGC
TTTTT TGTAAA.GT GTACAAA.T GT GATATAA.GAT T GGIT C T GC T.ACAT T CAGT TT C
TATAAAA.G T
GGT T C TAAAA.TAT TGTA.0 T G T CAA.T CAT C T CAT G.AT TA.T
TCTACTGTA.CACATTACTG.ACTITG
TAT GTAATAA.T TAATA.T TAGAAGA_AAA.TAT.AA.T T TAT II GAATAIAAAAAAAAAA
105241 SEQ ID NO: 46:
X iASLPX2LQX3E SVFQS GAHAYRI PALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAX4
T HQVQWQAQEVVAQARLDGHRSMNPCPLYDX5QT GT L FL FF 'AI P GQVTEQQQLQ T RANVTRLCQ
S TDHGRTWS S PRDL TDAAT GPAYREWS T FAVGPGHCL QLHDRARS LVVPAYAYRKLHPX6QRP
I P SAFC FL S H DHGRTWARGH FVAQDT LE C QVAEVE T GE QRVVT LNARS H LRARVQA Q S
TNDGLD
FQESQLVKKLVEPPPX7GCQGSVI S FP S PRS GPGSPAQWLLYTHP T HX 8X5QRADLGAYLNPRP P
A.PEAWSEPVLLAKGSX10AYSDLQSMGTGPDGSPLFGCLYEA.NDYEE IX11FX12MFTLKQAFPA.E
YLPQ
151
CA 03173557 2022- 9- 27
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105251 SEQ ID NO: 47:
XiX2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEHAELIVX9RRGD
YDAX10 THQVQWX ilAQEVVAQAX17LDGHRSMNPCPLYDX 13Q1 G TL FL FFIA_I PX14X15VTEX
16Q
QLQTRANVTRLX17X loVT STDHGRTWS SPRDLTDAAI GPX19YREWST FAVGPGHX20LQLHDRX21
RS LVVPAYAYRKLI¨IPX22QRP I P SAFX23 FL S HDHGRTWARGE FVAQDTX24E CQVAEVE TGEQRV
VT LNARSHLRARVQAQSX25NX26GLDFQX27S QLVKKLVE PPPX28GX29QGSVIS FPS PRSGPGS P
AQX30LLYTHPTHX31X22QRADLGAYLNPRPPAPEAWSEPX33LLAKGSX34AYSDLQSMGTGPDGS
PL FGX35LYEANDYEE IX36FX37MFTLKQ.AFP.AEYLPQ
105261 SEQ ID NO: 48:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDE QT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAA.I GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SA.FC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FPS PRSGPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAA.YSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQ
105271 SEQ ID NO: 49:
DAS L PYLQDE SVFQSGA.HAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAPTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDE QT GT L FL FFIAI PGQVTE QQQLQTRANVT RLCQVT
S TDHGRTWS S PRDLTDAA.I GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SA FC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PA.QWLLYTHPTHSWQRA.DLGAYLNPRPPAPEA.
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IRFIMFTLKQAFPAEYLPQ
105281 SEQ ID NO: 50:
DAS L PYLQKE SVFQSGA.HAYR I PALLYL P GQQS LLA.FAE QRASKKDEHAEL IVLRRGDYDANTH
QVQWQ.AQEVVAQARLDGHRSMNPCPLYDAQT GT L FL FFIA.I PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAA.I GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRA.RVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA.
WSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEE IVFLMFTLKQAFPAEYLPQ
105291 SEQ ID NO: 51:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAPTH
QVQWQ.AQEVVAQARLDGHRSMNPCPLYDAQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDA.A.I GPAYREWS T FAVGPGHCLQLHDRA.RSLVVPAYAYRKLHPKORP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPAGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHRKQRADLGAYLNPRPPAPEA
W SE PVLLAKGSA_AYSDLQSMG TGPDGS PL FGCLYE.ANDYEE IVFLMFTLKQAFPAEYLPQ
105301 SEQ ID NO: 52:
DAS L PYLQKE SVFQSGA.HA.YR I PALLYL P GQQS LLA.FAE QRASKKDEHA.EL IVLRRGDYDAS
TH
QVQWQAQEVVAQARLDGHRSMNPC PLYDAQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDL TDAA.I GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
152
CA 03173557 2022- 9- 27
LZ -6 -ZZOZ LSSELT0
ES;1,
IAODTHIANV2=1I0q000EIA05dIVIZZqZgISIOV=a3dNITAIS21HSCFIEVOVAA0VOMOAC
HIAVGAGS=ALIHVEE=ISVUOHVZV=S009T-LUYIVdIEAVHVOSOJASHNOgA.drISVG
:SS :ON CR OgS [9S01
C17
OTTA2VddVOHJIZI4IJAIZEXCENVEAq3aT-IdS0GdDIONS0qCSAVV=VqqAdES
M-VMTV-JdNENTKVOgGV-230MSHIdHIX7qMOVdS5d5S2JaSJJSIASSOD5OdddRATDDIATIOS
EaKFISCNISOVOA2IV2JgHSHVN'ILIAA2:10EDIEAEVA03EgIGOVAZHMJVADISHCHS'LEDAV
$aId240Ndl-FDDIRVX-VaAATIMIVNOWInFIDH5d5A-V3ISMX-Vd-DIVVCIERINdSSMI240HCI
SIAODTHIANVHICTIOn07IA00dIVIZZqZgISIOVGATIdDdNWS271HDCFIHVOVAA7OVOMOA 017
01-1IdVGX(192D7TIAIg7VINSVTIO7V1qVgq9000-1-VMI/W1-1V5SOJAS'3MOTRd'ISV
:LC :ON al Ogs Iscsol
OdqXV(33-VON'TITATIJAIHRAXINVRATID537JSCaDIDTATSCTIGSX_VDSONVq7AdHSM
-VEd-V-dd-adNTKV-D'IatT2i0MSHIdHIArYIMO-VdSSdDS-2:1dSddSIASSODSOdddaNDIHA'IOSE
SE
03=DGNISOVOA)I=ESEVNEIIAKHOEDIEAHVA03=IGOVAZHO)TdMDISHCHSEIZDZVS
ELDIOHdl-FDDiAVXVdANISH-V=Igng9H5dSAVZISME.21XVdSIVVGLYITddSSMIHSHCEIS
LIAOD'IELLANV2II0'100n2LIAODdraTI,43q3gISIO-VUXqd3dMIAIS2=1HOCFIEVOVAA2OVOMOAC
III(JVCAGS=ALIEVEEGMMSV-d0HVE=S009=qqVdIHAVHVSSOdASE>10qAdqSVIAI
:9S :ON GI OHS itS01
OE
OTTA2VddVON'ILL,EATIJAIZEXCENVE=35Z-IdS5CdDIONSCFRISAVDSDNVqqAdaS
MVEdVdd.>=1cENTKV-DgCV-210MSHIdHIX=MOVdS0dOS?JaSd3SIASOODDaddd2=DIAgOS
Eadd'ISCNISOVOA-2:1V-EgHSEV=AA2:10ESIEAEVAODEgI(10-V-AZHadVADISHCHS'LLDA-V-
SaI(DIONdl-Fal2=XVdAATISUVUGIT-10q3H9d9AVZISMEUXVdDIVVCIERaldSSMIEDHCI sz
SIAODTHIANV2iI0gOnnIAOSdIVIAA'1AgISIOVGATIdDdNWS-271HSG'IHVOVAA0VOMOA
01-1IdVGXCES2DITNIgHVIS-V210aV3VggS000dT=VM1VHVOSO3AS2NOTRd'ISV
:CC :ON ca Oas Iccgol
odqX2Vd3VONgI,31ATIZAIH2AGNVEAGOaqqdSOCdSIONSOgGSVVSONVqqAdHSM OZ
VEdVdd-HdNTA.VDEIGV-210HHIdHLATLIMOVdS0dDaldSdJSIAS5035VdddEAraIMATIOSZ
03(7-10GNISoVON2IVWIFIS2TVNgIAA-2105IAHVAO=IgnVA3P1521VMIUSHOHS73a4VS
dIdEOHd=1EAVXVdANISEV2KEWICTI3H5d5AVZISME21AVd5IVVGLYFDIdSSMIESHCEIS
LIAOD'IELLANV2IICTIDOOEIAODdraTIZZ'LdgIDIOVCIATIdDdHIAIS-2:114SCF-
DIVOVAA2OVOMOAC
HINVGXCEM:DigALIHV=MSVNOR-V,TV-qq9005drIXqq-VdDIXVH-VSSOAASNCTIAd'ISVO SI
:17'S :OK Cif Ogs Luso]
OdgiVa3VON'TITATIJAIHRAXINVRATID937dS9CaDITATSCTICSKYVSONVq7AdHSM
VEd-V-dd-adNTA:V=T230>DIHIdHiArLIMOVdSSdDS-2:1dSddSIASSODS-VdddaNDIXA'IOSE 01
03(TIOGNISOVOAUV=S?Td-MgIAA)10EDIEAHVA03=a0VAZHDWdMIUDHCHSEIZDZVS
dIdEOHd=1EAVXVdANISH-V=qng3H5d5AVZISMEEXV-dSIVVal=ddSSMIHSHCEIS
IA03THIANV-21I0q00nEIA00dIVI,33q3gISIOVCXTE3dNITAISTIHS=EVOVAA0VOMOA0
HILVGAGS2DYIALIEVEEGMMSV-2:10HVE=S009T-1=VdIEAVHVSSOdASENC=dqSVG
:ES :0N01 Os Ingo]
OdqXH-V-d3VOWIIZWgZAIHEACENVEAq3aqqdSS(IdDIDNSCTIC=VSSNVqqAdHSM
VEdVdd)IdN'IXVOgGV2IONEHIdHIXqqMOVdS0d5S2IdSdZSIASSODSVdddHAq?DlAgOSE
tOSITO/ZZOZSIVIDcl IZSOSI/ZZOZ OA%
WO 2022/150521
PCT/US2022/011504
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEA_NDYEE IVFLMFTLKQAFPAEYLPQ
105371 SEQ ID NO: 59:
DAS L PYLQKE SVFQSGAHAYR PALLYL P GQQS LLA FAE QRASKKDEHAEL IVLRRGDYDAPTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDAQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
S_AFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGS CAYSDLQSMGTGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQ
105381 SEQ ID NO: 60:
AASLPYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAPTH
QVQWQAQEVVAQARLDGI-IRSIV1NPCPLYJDAQTGTLFLFFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQ
105391 SEQ ID NO: 61:
MASLPYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAPTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDAQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARS LVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQ
105401 SEQ ID NO: 62:
AASLPYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAPTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDAQT GT L FL FFIAI PGQVTEQQQLQTRA_NVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCOGSVI S FP S PRS GPGS PAQWLLY THP THSWQRADLGAYLNPRP PAPE A
W SE PVLLAKGS CAYSDLQSMGTGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQ
105411 SEQ ID NO: 63:
EVQLLESGGGLVQPGGSLRLSCAASGFT FS SYIMMWVRQAPGKGLEWVS S I YPS GG I T FYADTV
KGRFT ISRDMSKNTLYLQMNSLPAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSAS TKGPSV
FPLAP S SKS T S GGTAALGCLVEDY FPE PVTVSWNS GALT SGVHT FPAVL QS SGLYSLS SVVTVP
S SSLGTQTYI CNVNHKP SNT KVDKKVE PKS CDKTHT CPP C PAPELLGGP SVFL FP PKPKDTLMI
S RT PEVTCVVVDVSHE DPEVK FNWYVDGVEVHNAKTKPREE QYNS TYRVVSVLTVLHQDWLNGK
E YKCKVSNKAL PAP I EKT I SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPS D IAVEW
E SNGQPENNYKT TPPVLDS DGS FFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLS P
GK
154
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[0542] SEQ ID NO: 64:
QSALTQPASVS GS PGQS IT IS CT GT S S DVGGYNYVSWYQQHPGKAPKLM I YDVSNRP S GVSNRF
SC-ISKSGNTASLT SC-ILQAEDEADYYCSSYTSSS TRVFC-ITGTKVTVLGOPKANPTVTLFPPSSEE
LQANKATLVCL I S DFYPGAVTVAWKADGS PVKAGVE T TKP SKQSNNKYAAS S YLS L T PE QWKS H
RS YS CQVTHE GS TVEKTVAP TECS
[0543] SEQ ID NO: 65:
QSALTQPASVS GS PGQS IT IS CT GT S S DVGGYNYVSWYQQHPGKAPKLM I YDVSNRP S GVSNRF
S GSKSGNTASL T I SGLQAEDEADYYCSSYTSSS TRVFGT GTKVTVLGQPKANPTVTL FP P S SEE
LQANKATLVCL I S DFYPGAVTV.AWKADGS PVKAGVE T TKP SKQSNNKY.AAS S YLS L T PE QWKS
H
RS YS CQVTHE GS TVEKTVAP TECS
[0544] SEQ ID NO: 66:
EVQLLESGGGLVQPGGS LRL S CAAS GET FS S YIMMWVRQAPGKGLEWVS S I YPS GG I T FYADTV
KGRFT I SRDNSKNTLYLQMNS LRA.EDTAVYYCARIKLGTVT TVDYWGQGTLVTVS SAS TKGPSV
FPLAP S SKS T S GGTAALGCLVKDYFPE PVTVSWNS GALT SGVHT FPAVL QS S GLYS L S
SVVTVP
S S S LGTQTY I CNVNHKP SNTKVDKKVE PKS CDKTHTCPPC PAPELLGGP SVFL FPPKPKDTLMI
SRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTI S KAKGQPRE PQVYTL PP SREEMTKNQVS LYCLVKGFYP S D IAVEW
ESNGQPENNYKT TPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GK
105451 SEQ ID NO: 67:
DAS L PYLQKE SVFQSGA.HAYR I PALLYL P GQQS LLA.FAE QRASKKDEHA.EL IVLRRGDYD.AGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDE QT GIL FL FFIAI PGQVTEQQQLQTRA.NVTRLCQVT
S TDHGRTWS S PRDLTDAA.I GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
ESQLVKKLVEPPPQGCQGSVI S FP S PRS GPGS PAQWLLY THP THSWQRADLGAYLNPRP PAPE A
W SE PVLLAKGSAAYSDLQSMGTGPDGS PL FGCLYE.ANDYEE IVFLMFTLKQAFPAEYLPQGGGG
S GGGGSDKTHT CPPCPAPE L LGGP SVFL FPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKAL P.AP I EKT I SKAKGQ
PRE PQVYTL P P SREEMIKNQVSLICLVKG FYP S D IAVEWE SNGQPENNYKT TPPVLDSDGSFFL
T S KL TVDKS RWQQGNVFS CS VMHEALHNHYT QKS LS LS P GK
[0546] SEQ ID NO: 68:
DAS L PYLQDE SVFQSGAHAYR PALLYL P GQQS LLA FAE QRASKKDEHAEL IVLRRGDYDAPTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDEQT GIL FL FFIAI PGQVTEQQQLQTRA.NVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SA.FC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
ESQLVKKLVEPPPQGCQGSVI S FP S PRS GPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMGTGPDGS PL FGCLYE.ANDYEE IRFIMFTLKQAFPAEYLPQGGGG
S GGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQ
PRE PQVYTL P P SREEMIKNQVSLICLVKG FYP S D IAVEWE SNGQPENNYKT TPPVLDSDGSFFL
T S KL TVDKS RWQQGNVFS CS VMHEALHNHYT QKS LS LS P GK
155
CA 03173557 2022- 9- 27
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105471 SEQ ID NO: 69:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLA.FAE QRA.SKKDEHA.EL IVLRRGDYD.ANTH
QVQWQAQEVVAQARLDGHRSIVINPCPLYDAQTGTLFLFFIAI PGQVTEQQQLQTRANVT RLCQVT
S TDHGRTWS S PRDLTDAA.I GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HL RARVQAQ S TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PA.QWLLYTHPTHSWQRADLGAYLNPRPPAPEA.
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQGGGG
S GGGGSDKTH T CP PCPAPE L LGGP SVFL FP PKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQ
PRE PQVYT L P P SREEMTKNQVSLTCLVKG FYP S D AVEWE SNGQPENNYKT TPPVLDS DGS FFL
T S KL TVDKS RWQQGNVFS CS VMHEALHNHYT QKS LSLSP GK
105481 SEQ ID NO: 70:
DAS L PYLQKE SVFQSGA.HAYR I PALLYL P GQQS LLA.FAE QRASKKDEHAEL IVLRRGDYDAPTH
QVQWQ.AQEVVAQ.ARLDGHRSMNPCPLYDAQT GT L FL FFIA.I PGQVTEQQQLQTRA.NVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPI P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPAGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHRKQRADLGAYLNPRPPAPEA.
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQGGGG
S GGGGSDKTH T CP PCPAPE L LGGP SVFL FP PKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQ
PRE PQVYT L P P SREEMIKNQVSLICLVKG FYP S D I.AVEWE SNGQPENNYKT TPPVLDS DGS FFL
T S KL TVDKS RWQQGNVFS CS VMHEALHNHYT QKS LSLSP GK
105491 SEQ ID NO: 71:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAS TH
QVQWQAQEVVAQARLDGHRSMNPGPLYDAQT GT L FL FFIAI PGQVTEQQQLQTRA.NVTRLCQVT
S TDHGRTWS S PRDLTDAA.I GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HL RARVQAQ S TNDGLDFQ
E SQLVKKLVE PPPAGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHRKQRADLGAYLNPRPPAPEA.
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYE.ANDYEE IVFLMFTLKQAFPAEYLPQGGGG
S GGGGSDKTH T CP PCPAPE L LGGP SVFL FP PKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQ
PRE PQVYT L P P SREEMIKNQVSLICLVKG FYP S D IAVEWE SNGQPENNYKT TPPVLDS DGS FFL
T SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
105501 SEQ ID NO: 72:
DA.SLPYLQKE SVFQSGA.HAYR I PALLYL P GQQS LLAFAE QRA.SKKDEHAEL IVLRRGDYDAT TH
QVQWQAQEVVAQARLDGHRSMNPCPLYDAQT GT L FL EFTA' PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPAGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHRKQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQGGGG
S GGGGSDKTH T CP PCPAPE L LGGP SVFL FP PKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQ
PRE PQVYT L P P SREEMTKNQVSLT CLVKG FYP S D IAVEWE SNGQPENNYKT TPPVLDS DGS FFL
T SKLT VDKS RWQQGNVFS CS VMHE.ALHNHYT QKS LSLSP GK
156
CA 03173557 2022- 9- 27
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[0551] SEQ ID NO: 73:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLA.FAE QRA.SKKDEHA.EL IVLRRGDYD.ANTH
QVQWQAQEVVAQARLDGHRSIVINPCPLYDAQTGTLFLFFIAI PGQVTEQQQLQTRANVT RLCQVT
S TDHGRTWS S PRDLTDAA.I GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVV TLNARSHLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPAGCQGSVI S FP S PRS GPGS PA.QWLLY THP THRKQRA.DLGAYLNPRP PAPEA.
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQGGGG
S GGGGSDKTH T CP PCPAPE L LGGP SVFL FP PKPKDT LMI SRTPEVICVV-VDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQ
PRE PQVYT L P P SREEMTKNQVSLTCLVKG FYP S D AVEWE SNGQPENNYKT TPPVLDS DGS FFL
T S KL TVDKS RWQQGNVFS CS VMHEALHNHYT QKS LSLSP GK
[0552] SEQ ID NO: 74:
X iASLPX2LQX3ESVFQSGAHAYRI PALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYD1\X4
T HQVQWQAQEVV.AQARLDGHRSMNPCPLYDX5QT GT L FL FF IAI P GQVTEQQQLQ T RANVTRLCQ
VT S TDHGRTWS S PRDLTDAAIGPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPX6QRP
I P SAFC FL S HDHGRTWARGH FVAQDT LE C QVAEVE T GE QRVVT LNARS HLRARVQAQS
TNDGLD
FQESQLVKKLVEPPPX7GCQGSVI S FP S PRS GPGSPAQWLLYTHP THX8X9QRADLGAYLNPRPP
A_PEAWSEPVLLAKGSX10AYSDLQSMGTGPDGS PLFGCLYEANDYEE IX11FX12MFTLKQAFPAE
YLPQGGGGS GGGGSDKTHT C P PCPAPELL GGP SVFL FPPKPKDT LMI SRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHODWLNGKE YKCKVSNKA.L PAP I EK
T I SKAEGQPRE PQVYT LPPS REEMTKNQVS L T CLVKG FY P S D IAVEWE SNGQPENNYKT
TPPVL
DS DGS FFL T S KL TVDKSRWQQGNVFS CSVMHEALHNHYT QKS L S LS PGK
[0553] SEQ ID NO: 75:
X1X2SX3X4X5U2X6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEH_AELIVX9RRGD
YDAX10 THQVQWX 11.A.QEVVAQAX12LDGHRSMNPCPLYDX 13QT G T L FL FFIAI
PX14X15VTEX16Q
QLQTRANVTRLX17Xi8VT STDHGRTWS SPRDLTDAA_I GPXigYREWST FAVGPGHX20LQLHDRX7j.
RS LVVPAYAYRKLHPX22QRP I P SAFX23 FL S HDHGRTWARGH FVAQDTX24E CQVAEVE T GEQRV
VT LNARSHLRARVQAQSX25NX26GLDFQX27S QLVKKLVE PPPX28GX29QCSVIS FPS PRSGPGS P
A_QX3oLLYTHPTHX3iX32QRA_DLGAYLNPRPPAPEAWSEPX33LLAKGSX34AYSDLQSMGTGPDGS
PL FGX3sLYEANDYEE IX36FX37MFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGG
P SVFL FPPKPKDT LMI SRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYR
VVSVL TVLHQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQPREPQVYTLPPSREEMTKNQVSL
TCLVKGFYPS D IAVEWESNGQPENNYKT T PPVLDSDGS F FL T SKLTVDKSRWQQGNVFS CSVMH
EALHNHYTQKS L S LS PGK
[0554] SEQ ID NO: 76:
CAA.TCTGCTCT TACACAGCC T GCCAGCGT GT CCGGATCT CC T GGCCAGAGCATCACCAT CAGC T
TACCGGCACCAGCTC T GAT GTCGGCGGC TACAA.T TACG T GT CCTGGTAT CAGCAGCACCCCGG
CAAGGCCCC TAAGCTGA T GA_T CTACGACG T GT CCAACAGACCCAGCGGC GT =CAA TAGAT IC
T CCGGCAGCAA GAGCGGCAA_CAC C GC C AG C C T GACAA T TAGCGGACT GCAGGCCGAGGACGAGG
CCGAT TAC TA_C T GTAGCAGC TACA CCAGC T CCAGC AC CAGAGT GT T T GGCAC
CGGCACAA_AAGT
GACCGTGC T GGGCCAGCC TA.AGGCCAA.T CC TACCGT GACAC T GT TCCCTCCAAGCA.GCGAGGAA.
C T GCAGGC TAA.CAAGGCCACACT CGT GT GCC T GAT CAGCGAC T T T TAT CC T GGCGCCG T
GACCG
T GGCC TGGAAGGC TGAT GGAT CT CCAGT GAA_AGCCGGCG T GGAA_ACCAC CAA.GCC TAGCAAGCA
GAG CAA.CAA.CAA_ATAC GC C G C CAG CAGC TACC T GAGCCT GACAC C T GAG CAG TGGAA.G
T CCCAC
157
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AGATCCTACAGCT GCCAAGT GAC C CAC GAGGGCAGCACC G T GGAAAAAACAGTGGCCCC TACCG
AGT GC TCT
105551 SEQ ID NO: 77:
GAGGT GCAGC T GC TGGAAT C T GGCGGAGGAC =GT TCAGCC T GGCGGC T C TC TGAGAC T GTC
T T
GT GCCGCCAGCGCCTICACC T TCAGCAGC TATATCATGAT GT GGGTCCGACAGGCCCC T GGCAA
AGGCCITGAATGGGIGICCAGCATCTATCCCAGCGGCGGCATCACCTT T TACGCCGACACAGTG
AAGGGCAGAT TCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCC
TGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAGAATCAAGCTGGGCACCGTGACCACCGT
GGAT TAT T GGGGACAGGGCACCCT GGTCACCGT GTCATC T GC TAGCACCAAGGGCCCAT CCGTC
T T CCCCCT GGCACCCT CC T C CAAGAGCAC CT CT GGGGGCACAGCGGCCC T GGGCT GCC T GGT
CA
AGGACTACTTCCCCGAACCGGTGACGGTGICCIGGAACTCAGGCGCTCTGACCAGCGGCGTGCA
CACC T TCCCGGC T &ICC TACAGTCC TCAGGACTC TAC TCCC TCAGCAGCGT GGT GACCGT GCCC
TCCAGCAGCT TGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG
T GGACAAGAAAGT TGAGCCCAAATC T T GT GACAAAAC TCACACATGCCCACCGT GCCCAGCAC C
T GAAC TCC T GGGGGGACCGT CAGT CT T CC TC T T CCCCCCAAAACCCAAGGACACCC T CAT
GATC
TCCCGGACCCCTGAGGICACATGCGTGGTGGIGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
T CAAC T GG TA_C G T GGAC GGC G T GGAGG T G CATAAT GC CAAGACAAAGC C GC
GGGAGGAG CAG TA
C_AACAGCACGTACCGT GT GGTCAGCGTCC TCACCGTCCT GCACCAGGAC T GGCT GAA T GGCAAG
GAG TACAAGT GCAAGGIC T CCAACAAAGCCC TCCCAGCCCCCATCGAGAAAACCATC T CCAAAG
CCAAAGGGCAGCCCCGAGAACCACAGGTC TACAC C C T GC C C C CAT CC C G GGAGGAGAT GACCAA
GAACCAGGTCAGCCIGTACTGCCIGGICAAAGGCTICTATCCCAGCGACATCGCCGTGGAGIGG
GAGAGCAAT GGGCAGCCGGAGAACAAC TACAAGAC CACGCC TCCCGT GC T GGACTCCGAC GGC T
CC T TC T TCC T C TATAGCAAGC TCACCGT GGACAAGAGCAGGT GGCAGCAGGGGAACGT C T TC TC
AT GC T CCGT GAT GCAT GAGGC TCT GCACAACCAC TACAC GCAGAAGAGCC TC TCCC T GT C
TCCG
GGTAAA
105561 SEQ ID NO: 78:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDEQT GIL FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
S OT,VT<KT ,VE P PPOGCOGSVT S FPS PR S C_1PGS PAOWT ,T.Y T-FTP THSWOR ADT,GAYT
NPR P PAPF. A
W SE PVLLAKGSAAYSDLQSMGTGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQGGGG
SDKTHTCPPCPAPELLGGPSVFLFPPKPKDILMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLTSKLT
VDKSRWQQGNVFS CSVMHEALHNHYTQKS L S LS PGK
105571 SEQ ID NO: 79:
GAT GCATC TC T GCCTTACC T GCAGAAAGAAAGCGT GTTCCAGTC TGGCGCCCACGCC TACAGAA
T TCCCGCTC T GC T GTATC T GCCAGGCCAGCAGTC TC TGC T GGC T T TCGC T
GAACAGCGGGCCAG
CAAGAAGGAT GAGCAC GC C GAAC T GAT C G T GC T GC GGAGAGGC GAT TAC GAC GC C
GGCACACAT
CAGGT GCAGT GGCAGGC TCAAGAGGT GGTGGC TCAGGCTAGAC T GGACGGCCACAGAT C TAT GA
A_CCCCTGICCTCTGTACGATGAACAGACCGGCACACTGTT TC T GTTC T T TATCGC TAT CCCCGG
CCAAGTGACCGAGCAGCAGCAGCTGCAGACAAGAGCCAACGTGACCAGACTGTGTCAAGTGACC
TCCACCGACCACGGCAGAACCIGGICTAGCCCTAGAGATCTGACCGACGCCGCCATCGGACCTG
CC TATAGAGAG T GGTCCACC T TCGCCGT T GGACC T GGACAC T GI CTCCAGC T GCACGACAGGGC
TAGA TC TC T GGT GGTGCC T GCCTACGCC TA TAGAAAGCT GCACCCCAAACAGCGGCC T AT TCC T
AGCGCC TIC T GC T TIC GAGCCACGATCACGGCAGGACAT GGGCCAGAGGACATT TCGT GGCCC
158
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AGGACACAC T GGAATGCCAGGIGGCCGAAGIGGAAACCGGCGAGCAGAGAGT CGT GACCC TGAA
C GCCAGAT C T CACCTGAGAGCCAGAGT GCAGGCCCAGAG CACAAACGAC GGCCT GGAT T TCCAA
GAGAGCCAGC T GGICAAGAA_ACT GGT GGAACC T CC T CCACAGGGCTGT CAGGGAAGCG T GAT CA
GCTTTCCATCTCCTAGAA_GCGGCCCTGGCTCTCCTGCTCA_GTGGCTGCTGTATACACACCCCA_C
ACACAGCT GGCAGAGAGCCGATCT GGGCGCC TACC T GAAT CC TAGACC T CC T GCT CC T GAGGC
T
T GGAGCGAAC C T GT TC T GC T GGCCAAGGGCAGCGC T GCC TACAGCGAT C T GCAGT C TAT
GGGCA
C_AGGCCCT GAT GGCAGCCC T C TGT ITGGCTGICTGTACGAGGCCAACGACTACGAAGA_GATCGT
GT T CC TGAT G T T CACCC T GAAGCAGGCCT T TCCAGCCGAGTACCTGCCTCAAGGCGGAGGCGGA
T CCGACAAAAC T CACACAT GCCCACCGT GCCCAGCACCT GAAC T CCT GGGGGGACCGT CAGTC T
TCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT
GGT GGTGGAC G T GAGCCACGAAGACCC T GAGGTCAAGT T CAAC T GGTAC GT GGACGGC G T
GGAG
G T GCATAAT GCCAAGACAAAGCCGCGGGAGGAGCAG TACAACAGCACGTACCGT GT GG T CAGCG
TCCT CAC CGTCCTG CAC CAG GAC T GGC T GAAT GGCA_AGGAGTACAAGT G CAAGG TCTC
CAACAA
A_GCCC T CC CA_G C C CCCA T C GAGAA_AAC CAT C T CCAAAGC CAAAGGGCA_G CCCC GA
GAAC CACAG
GTCTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGG
TCAAAGGCT T C TATCCCAGC GACATCGCC GT GGAGTGGGAGAGCAAT CGGCAGCCGGAGAACAA
C TACAAGACCACGCCT CCCG T GCT GGAC T CCGACGGC T CC T TCT TCC T CAC TAGCAAGC T
CACC
GT GGACAAGAGCAGGT GGCACCAGGGGAACGT C T IC TCAT GC T CCGT GAT GCAT GAGGC T CT
GC
ACAAC CAC TACAC GCAGAAGAGCC IC T CCC T GT C T CCGGG TAAA
105581 SEQ ID NO: 80:
GAT GCATC T C T GCCTTACC T GCAGAAAGAAAGCGT GIT CCAGT C TGGC GCCCACGCC TACAGAA
T T CCCGCT C T GC T GTAT C T GCCAGGCCAGCAGT C T C TGC T GGC T
TTCGCTGAACAGCGGGCCAG
CAAGAAGGAT GAGCAC GC C GAAC T GAT CG T GC T GC GGAGAGGC GAT TAC GAC GC C
GGCACACAT
CAGGT GCAGT GGCAGGC T CAAGAGGT GGT GGC T CAGGCTAGAC T GGAC GGCCACAGAT C TAT GA
A_CCCC TC-IT CC T C T GTACGAT GAACAGACC GGCACAC TC-1T TTCT GTTC T T TAT CGC
TAT CCCCGG
C CAAG T GAG C GAG CAG CAG CAGC T GCAGACAAGAGCCAACGT GACCAGAC T G TGT CAAG T
GACC
TCCACCGACCACGGCAGAACCTGGTCTAGCCCTAGAGATCTGACCGACGCCGCCATCGGACCTG
CC TATAGAGAG T GGTCCACC T TCGCCGT TGGACCTGGACAC T GT CTCCAGC T GCACGACAGGGC
TA_GAT C TC T GG T GGTGCC T GCCTA_CGCC TATAGAAA_GCT GCACCCCAAACAGCGGCC TAT
TCCT
AGCGCC TTCT GC T TTCTGAGCCACGATCACGGCAGGACATGGGCCAGAGGACATT TCGTGGCCC
AGGACACACTGGAATGCCAGGTGGCCGAAGTGGAAACCGGCGAGCAGAGAGTCGTGACCCTGAA
CGCCAGATCTCACCTGAGAGCCAGAGTGCAGGCCCAGAGCACAAACGA_CGGCCIGGAT T T CC AA
GAGAGCCAGC T GGTCAAGAAACT GGT GGAACC T CC T CCACAGGGCTGT CAGGGAAGCG T GAT CA
GC T T T CCAT C T CC TAGAAGC GGCCCT CGC T C T CC T GC T CAGT CGCTGC T
GTATACACAC CCCAC
ACACAGCT GGCAGAGAGCCGATCT GGGCGCC TACC T GAAT CC TAGACC T CC T GCT CC T GAGGC
T
T GGAGCGAACC T GT TC T GC T GGCCAAGGGCAGCGC T GCC TACAGCGAT C T GCAGTC TAT
GGGCA
CAGGCCCT GAT GGCAGCCC T C TGT IT GGC T GTCT GTACGAGGCCAACGAC TACGAAGAGATCGT
GTTCCTGATGT TCACCCTGAAGCAGGCCITTCCAGCCGAGTACCTGCCTCAA
105591 SEQ ID NO: 81:
DAS L PYLQKE SVFQSGAHAYR PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDEQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVEPPPQGCQGSVI S FP S PRS GPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQEPKS
S DKTHTCP PC PAPELLGGP SVFL FPPKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYNS T YRVVSVL TVLHQDWLNGKE YKCKVS NKAL PAP I E KT I S KAKG Q PRE
P Q
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VYTLPPSREEMTKNQVSLTCLVEGFYPS D IAVEWESNGQPENNYKTTPPVLDSDGS FEL TSKLT
VDKSRWQQGNVFS CSVMHEALHNHYT QKS L S LS PGK
105601 SEQ ID NO: 82:
DAS L PYLQDE SVFQSGAHAYR PALLY', P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAPTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDEQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAT GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE I RF IMFT LKQAFPAEYL P QEPKS
S DKTHTCP PC PAPELLGGP SVFL FPPKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKT ISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPS D IAVEWESNGQPENNYKTTPPVLDSDGS FEL TSKLT
VDKSRWQQGNVFS CSVMHEALHNHYT QKS L S LS PGK
105611 SEQ ID NO: 83:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKEDEHAEL IVLRRGDYDANTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDAQT GT L FL FFIAI PGOVTEQQQLOTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQEPKS
S DKTHTCP PG PAPELLGGP SVFL FP PKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKT ISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPS D IAVEWESNGQPENNYKTTPPVLDSDGS FEL TSKLT
VDKSRWQQGNVFS CSVMHEALHNHYT QKS L S LS PGK
105621 SEQ ID NO: 84:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAPTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDAQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDI GPAYREWS T FAVGPGHCLQLHDRRSLVVPAYAYRKLHPKQRPI P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPAGCQGSVI S FPS PRS GPGS PAQWLLY THP THRKQRADLGAYLNPRP PAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQEPKS
S DKTHTCP PC PAPELLGGP SVFL FPPKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FEL TSKLT
VDKSRWQQGNVFS CSVMHEALHNHYT QKS L S LS PGK
105631 SEQ ID NO: 85:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAS TH
QVQWQAQEVVAQARLDCHRSMNPCPLYDAQICT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPAGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHRKQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGDDGS DL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQEPKS
S DKTHTCP PC PAPELLGGP SVFL FP PKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
160
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VYTLPPSREEMTKNQVSLTCLVEGFYPS D IAVEWESNGQPENNYKTTPPVLDSDGS FEL TSKLT
VDKSRWQQGNVFS CSVMHEALHNHYT QKS L S LS PGK
105641 SEQ ID NO: 86:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAT TH
QVQWQAQEVVAQARLDGHRSMNPCPLYDAQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDI GPAYREWS T FAVGPGHCLQLHDRRSLVVPAYAYRKLHPKQRPI P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPAGCQGSVI S FPS PRSGPGSPAQWLLYTHPTHRKQRADLGAYLNPRPPAPEA
W SE PVLLAKGSA_AYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQEPKS
S DKTHTCP PC PAPELLGGP SVFL FPPKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FEL TSKLT
VDKSRWQQGNVFS CSVMHEALHNHYT QKS L S LS PGK
105651 SEQ ID NO: 87:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDANTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDAQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPAGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHRKQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQEPKS
S DKTHTCP PC PAPELLGGP SVFL FPPKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
VYTLPPSREEMTKNQVSLTCLVKGFYPS D IAVEWESNGQPENNYKTTPPVLDSDGS FEL TSKLT
VDKSRWQQGNVFS CSVMHEALHNHYT QKS L S LS PGK
105661 SEQ ID NO: 88:
X iASLPX2LQX3ESVFQSGAHAYRI PALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAX4
T HQVQWQAQEVVAQARLDGHRSMNPCPLYDX5QT GT L FL FF IAI P GQVTEQQQLQ T RANVTRLCQ
VT S TDHGRTWS S PRDL T DAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPX6QRP
I P SAFC FL S HDHGRTWARGH F-VAQDT LE C QVAEVE T GE QRVVT LNARS HLRARVQAQS
TNDGLD
FQESQLVKKLVEPPPX7GCQGSVI S FP S PRS GPGSPAQWLLYTHP THX0X9QRADLGAYLNPRPP
APEAWSEPVLLAKGSX10AYSDLQSMGTGPDGS PLFGCLYEANDYEE IX11FX12MFTLKQAFPAE
YL PQX13DKTHT CP P CPAPELLGGP SVFL FP PKPKDTLMI SRT PEVTGVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQ
PRE PQVYT L P P SREEMIKNQVSLICLVKG FYP S D IAVEWE SNGQPENNYKT TPPVLDS DGS FFL
T S KL TVDKS RWQQGNVFS CS VMHEALHNIIYT QKS LS LS P GK
105671 SEQ ID NO: 89:
X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEFLAELIVX9RRGD
YDAX THQVQWX11AQEVVAQAX12LDGHRSMNPCPLYDX 13Q1 G TL FL FFIAI PX14X15VTEX16Q
QLQTR7ANVTRLX17X18VT STDHGRTWS SPRDLTDAAI GPX19YREWST FAVGPGHX2oLQLHDRX24
RSLVVPAYAYRKLHPX22QRPI P SAFX23 FL S HDHGRTWARGH FVAQDTX24E CQVAEVE TGEQRV
VT LNARSHLRARVQAQSX25NX26GLDFQX27S QLVKKLVE PPPX28GX29QGSVIS FPS PRSGPGS P
AQX3oLLYTHPTHX31X32QRADLGAYLNPRPPAPEAWSEPX33LLAKGSX34AYSDLOSMGTGPDGS
PL FGX35LYEANDYEE IX36FX37MFT LKQAFPAEYL PQX3EDKT H T CP PCPAPE LLGGP SVFL FPP
KPKDTLMI SRT PEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPRE E QYNS TYRVVSVLTVL
161
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HQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKT T PPVLDSDGS FFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK
105681 SEQ ID NO: 90:
GGGGSGGGGS
105691 SEQ ID NO: 91:
E PKS S
105701 SEQ ID NO: 92:
E PKS CDKTHT C PPCPAPELL GGPSVFL FP PKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNS T YRVVSVL TVLHQDWLNGKE YKCKVSNKAL PAP I EKT I SKAKGQP
RE PQVYTL PP SREEMTKNQVS LT CLVKGFYP S D IAVEWE SNGQPENNYKT TPPVLDSDGSFFLT
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
105711 SEQ ID NO: 93:
DKTHTCPPCPAPELLGGPSVELFPPKPKDILMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREE QYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAP I E KT I SKAKGQ PRE PQV
Y T L PP SREEMTKNQVS LYCLVKGFYP S D IAVEWE SNGQPENNYKTTPPVLDS DGS FFLYSKL TV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
105721 SEQ ID NO: 94:
DAS L PYLQKE SVFQSGAHAYR PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDEQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGSPAQWLLYTHPTHARQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQ
105731 SEQ ID NO: 95:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDE QT GT L FL FFIAI PGQVTEQQQLQTRA_NVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARS LVVPAYAYRKLHPKQRP I P
SAEC EL SHDHGRTWARGH PVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PAQWLLY THP THARQRADLGAYLNPRP PAPE A
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQGGGG
S GGGGSDKTH T CP PCPAPE L LGGP SVFL FP PKPKIYILMI SRTPEVICVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKAL RAP I EKT I SKAKGQ
PRE PQVYT L P P SREEMIKNQVSLICLVKG FYP S D IAVEWE SNGQPENNYKT TPPVLDSDGSFFL
T S KL TVDKS RWQQGNVFS CS VMHEALHNHYT QKS LSLSP GK
105741 SEQ ID NO: 96:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDEQT GT L FL FFIA PGQVTEQQQLQTRA_NVTRLCQVT
162
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S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PA.QWLLYTHPTHARQRADLGAYLNPRPPAPEA.
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEA_NDYEE IVFLMFT LKQAFPA_EYL P QEPKS
S DKTHTCP PC PAPELLGGP SVFL FPPKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
VYTLPPSREEMTKNQVSLTCLVKGFYPS D AVEWESNGQPENNYKTTPPVLDSDGS FEL TSKLT
VDKSRWQQGNVFS CSVMHEALHNHYT QKS L S LS PGK
105751 SEQ ID NO: 97:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQ.AQEVVAQARLDGHRSMNPCPLYDEQT GT L FL FFIA.I PGQVTEQQQLQTRANVTRLCYVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPTGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA.
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQ
[0576] SEQ ID NO: 98:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQ.AQEVVAQARLDGHRSMNPCPLYDEQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCYVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPI P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPTGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFT LKQ.AFPA.EYL P QGGGG
S GGGGSDKTH T CP PCPAPE L LGGP SVFL FP PKPKDITMI SRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQ
PRE PQVYT L P P SREEMIKNQVSLICLVKG FYP S D IAVEWE SNGQPENNYKT TPPVLDS DCS FFL
T SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0577] SEQ ID NO: 99:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDE QT GT L FL FFIAI PGQVTEQQQLQTRA.NVTRLCYVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T F.AVGPGHCLQLHDRARSLVVPAY.AYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LN.ARS HLRARVQAQS TNDCLDFQ
E SQLVKKLVE PPPTGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA.
W SE PVLLAKGSAAYSDLQSMC TGI'DGS PL FGCLYE.ANDYEE IVELMFTLKQ.AFFA.EYLPQEPKS
S DKTHTCP PC PAPELLGGP SVFL FPPKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
VYTLPPSREEMTKNQVSLTCLVKGFYPS D IAVEWESNGQPENNYKTTPPVLDSDGS FFL TSKLT
VDKSRWQQGNVFS CSVMHEALHNHYT QKS L S LS PGK
[0578] SEQ ID NO: 100:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDEQT GT L FL FFIAI PGQVTEQQQLQTRA_NVTRLCYVT
S TDHGRTWS S PRDLTDAA.I GPAYREWS T FA.VGPGITCLQLHDRARSLVVPAY.AYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLR FRVQAQS TNDGLDFQ
E SQLVKKLVE PPPTGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA.
W SE PVLLAKGSAAYSDLQSMC TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQ
163
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105791 SEQ ID NO: 101:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQ.AQEVVAQARLDGHRSMNPCPLYDE QT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCYVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
S_AFC FL SHDHGRT WARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLR FRVQAQS TNDGLDFQ
E SQLVKKLVE PPPTGCQGSVI S FP S PRS GPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQGGGG
S GGGGSDKTH T CP PCPAPE L LGGP SVFL FP PKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNS TYRVV SVL TVLHQDWLNGKEYKCKVSNKAL PAP EKT I SKAKGQ
PRE PQVYT L P P SREEMTKNQVSLTCLVKG FYP S D IAVEWE SNGQPENNYKT TPPVLDSDGSFFL
T S KL TVDKS RWQQGNVFS CS VMHEALHNHYT QKS LSLSP GK
105801 SEQ ID NO: 102:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDEQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCYVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVCPCHCLOLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLR FRVQAQS TNDGLDFQ
E SQLVKKLVE PPPTGCQGSVI S FP S PRS GPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVELMFTLKQAFPAEYLPQEPKS
S DKTHTCP PC PAPELLGGP SVFL FPPKPKDILMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
VYT L P P SREEMTKNQVS L T CLVKGFYP S D AVEWESNGQPENNYKTTPPVLDSDGS FFL TSKLT
VDKSRWQQGNVFS CSVMHEALHNHYT QKS L S LS PGK
105811 SEQ ID NO: 103:
GA_T GCATC T C T GCCTTACC T GCAGAAAGAAAGCGT GIT CCAGT C TGGCGCCCACGCC TACAGAA_
T T CCCGCT C T GC T GTAT C T GCCAGGCCAGCAGTC T C TGC T GGC T TTCGC T
GAACAGCGGGCCAG
C_AAGA_AGGAT GAG CAC G C C G_AAC T GAT CG T GC T GCG GAGAG G C GAT TA_C GAC GC
C G G CACACAT
CAGGT GCAGT GGCAGGC T CAAGAGGT GGT GGC T CAGGCTAGAC T GGAC GGCCACAGA T C TAT
GA
ACCCCTGTCCTCTGTACGATGAACAGACCGGCACACTGTTTCTGTTCTTTATCGCTATCCCCGG
CAGTGACCGAGCAGCAGCAGCTGCAGACGAGCCAZ\CGTGACCAGACTGTGTCALGTGACC
TCCACCGACCACGGCAGAACCTGGTCTAGCCCTAGAGATCTGACCGACGCCGCCATCGG_ACCTG
CC TATAGAGAG T GGTCCACC T TCGCCGT I GGACC T GGACAC T GT CTCCAGC T GCACGACAGGGC
TAGAT C IC I GG T GGTGCC T GCCTACGCC TATAGAAAGCT GCACCCCAAACAGCGGCC TAT TCCT
A_GCGCC T TC T GC T TIC I GAGCCACGAT CA_CGGCAGGACAT GGGCCAGA_GGACAT T
TCGTGGCCC
A_GGACACAC T G GAAT GC CAG G T GGCC GAAG T GGAAAC CG GC GAG CAGA_GAG T CGT GAC
C C TGAA
C GC CAGAT C T CAC C T GAGAG C CAGAG T GCAGGCCCAGAGCACAAACGAC GGCCT GGAT T T
CCAA
GAGAGCCAGC T GGTCAAGAAACT GGT GGAACC T CC T CCACAGGGCTGT CAGGGAAGCG T GAT GA
GC T T T CCAT C T CC TAGAAGCGGCCC T GGC T C TCC T GC T CAGTGGCTGC T
GTATACACA_CCCCAC
ACACAGCT GGCAGAGAGCCGATCT GGGCGCC TACC T GAAT CC TAGACC T CC T GCT CC T GAGGC
T
T GGAGCGAACC T GT TCTGC T GGCCAAGGGCAGCGC T GCC TACAGCGAT C T GCAGTC TAT GGGCA
CAGGCCCT GAT GGCAGCCC T C TGT TI GGC I GT C T GTACGAGGCCAACGAC TACGAAGAGATCGT
GT T CC TGAT G T TCACCCTGAAGCAGGCCT I I CCAGCCGAG TACC TGCC T CAAGGCGGAGGCGGA
TCCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCT
T CC T C T TCCCCCCAAAACCC_AAGGACACCC T CAT GATCT CCCGGACCCC T GAGGT CACAT GCGT
GGTGG'TGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACC4TC;CgAMC4C.CgTCgCgAg
GT GCATAAT GCCAAGACAAAGCCGCGGGAGGAGCAG TACAACAGCACGTACCGT GT GG T CAGCG
T CC T CACCGT CC T GCAC CAG GACT GGC T GAAT GGCAAGGAG TACAAGT GCAAGGTC T
CCAACAA
164
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AGCCC TCCCAGCCCCCAT CGAGAAAAC CAT C T CCAAAGCCAAAGGGCAGCCCCGAGAAC CACAG
GTCTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGG
T CAAAGGC T T C TAT CC CAGC GACAT C GC C GT GGAGT GGGAGAGCAAT GG GCAGC C
GGAGAACAA
C TACAAGACCACGCCT CCCG T GCT GGAC T CCGACGGC T CC T TCT TCC T CAC TAGCAAGC T
CACC
G T GGACAAGAGCAGGTGGCAGCAGGGGAACGT C T TC TCAT GC T CCGT GAT GCAT GAGGC T CT
GC
ACAAC CAC TACAC GCAGAAGAGCCTC T CCC T GTC T CCGGG TAAA
105821 SEQ ID NO: 104:
EVQLLESGGGLVQPGGSLRLSCAASGFT FS S YIMMWVRQAPGKGLEWVS S YPS GG T FYADTV
KGRFT I SRDNSKNTLYLQMNS LRAEDTAVYYCARIKLGTVT TVDYWGQG T LVTVS SAS TKGP SV
FPLAP S SKS T S GGTAALGCLVKDYFPE PVTVSWNS GALT SGVHT PPAVL QS S GLYS S SVVTVP
S S S LGTQTY I CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
S RT PEVTCVVVDVSHE DPEVK FNWYVDGVEVHNAKTKPREE QYGS TYRVVSVLTVLHQDWLNGK
E YKCKVSNKAL PAP I EKT I SKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALEINHYTQKSLSLSP
GK
105831 SEQ ID NO: 105:
GAGGT GCAGC T GC TGGAAT C T GGCGGAGGAC T T GT T CAGCC T GGCGGC TCTC TGAGAC T
GTC T T
G T GCCGCCAGCGGCT TCACC T TCAGCAGC TATAT CATGAT GT GGGTCCGACAGGCCCC T GGCAA
A_GGCCITGAATGGGIGTCflAGCATC!TATC.C.flAGC.GGrGGF!ATC.AMTT T TACIGMGArArAGTG
AAGGGCAGA.T TCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCC
TGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAGAATCAAGCTGGGCACCGTGACCACCGT
GGAT T.AT T GGGG.ACA.GGGCACCCIGGICACCGT GT C.AT C T GC T.AGCACCAA.GGGCCCAT
CCGT C
T T C. CT' T GGCACCCT CC T CCAAGA.GCACCTC T GGGGGCACAGCGGCCC T GGGCT GCC T
GGTCA
AGGAC T.AC T T C CCCGAACCGG TGACGGT G T CUT GGAACT CAGGCGCT C T GACCAGCGGC
GTGCA
CACC T TCCCGGC T GTCC TACAGT CC T CAGGACT C TAC IC CC T CAGCAGC GI GGT GACCG T
GCCC
TCCAGCAGCT T GGGCACCCAGACC TACAT C T GCAACGT GAAT CACAAGCCCAGCAACAC CAAGG
T GGACAAGAAAGT TGAGCCCAAAT CT T GT GACAAAAC T CACACATGCCCACCGT GCCCAGCAC C
T G.AAC TCC T GGGGGGA.CCGT C.AGICT ICC TC T TCCCCCCAAAACCCAAGG.ACACCC T CA.T
GAT C
TCCCGGACCCCTGAGGICACATGCGTGGTGGIGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
T CAAC TGGTACGT GGACGGCGTGGAGGT GCATAAT GC CAAGACAAAGC C GC GGGAGGAG CAG TA
CGGTAGCACG TACCGT GT GG T CAGCGT CC T CACCGT CCT GCACCAGGAC T GGCT G.AAT
GGCAAG
GAG TAC.AAG T G CAAGG TCT C CAACAAAGC CC T CC CAGCC C C CAT CGAG.AAAAC CAT C T
CCAAAG
CCAAAGGGCAGCCCCGAGAACCACAGGT C TACACCC T GC C C C C.AT CC C G GGAGGAGAT GACCAA
GAACCAGGT CAGCCIGTAC T GCCT GGTCAAAGGC T TC TAT CCCAGCGACAT CGCCGT GGAGTGG
GAGAGC.AAT GGGCAGCCGGAGAAC.AA.0 TAC.AAGA.0 CA.CGCC T CCCGT GC T GGACT CCGAC
GGC T
GC TTCT TCC T C TATAGCAAGC TCACCGT GGACAAGAGCAGGT GGCAGCAGGGGAACGT C T TC T C
AT GC T CCGT GAT GCAT GAGGC TCT GCACAACCAC TACACGCAGAAGAGCC T C TCCC TGTC TCCG
GGTAAA
105841 SEQ ID NO: 106:
TVEKSVVFK_AEGEHFTDQKGNT IVGS GS GGT TKYFRI PAMC T T SKGT IVVFADARHNTASDQS F
I DTAAARS T DGGKTWNKK 'AI YNDRVNS KL S RVMDP TC I VAN I QGRE T I
LVMVGKWNNNDKTWG
AYRDKAPDTDWDLVLYKS TDDGVT FSKVE TNI HD IVTKNG T I SAMLGGVGS GLQLNDGKLVFPV
QMVRTKNI T TVLNT S F I YS TDGI TWS L P S GYCEGFGSENN I I E FNA.S LVNNIRNS
GLRRS FE TK
D FGKTWTE FP PMDKKVDNRNHGVQGS TITIPS GNKLVAAHS SAQNKNNDYTRSDI SLYAFINLYS
GEVKL I DDFYPKVGNAS GAGYSCL SYRKNVDKE T LYVVYE.ANGS IE FQDL SRHLPVI KS YNGGG
GS GGGGSDKT H T GPPCPAPE LLGGPSVFL FP PKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNW
165
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YVDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHQDWLNGKE YKCKVS NKAL PAP I EKT I SKAKG
QPRE PQVYT L P P SREEMTKNQVS L TCLVKGFYP S D IAVEWE SNGQPENNYKT TPPVLDS DGS FF
L TSKLTVDKSRWQQGNVFSCSVMHEALFINHYTQKSLSLS PGKSGGGGSHHHHHHHH
105851 SEQ ID NO: 107:
A_CAGT GGAAAAGT CCGT GGT GTT CAAGGCCGAGGGCGAG CAC T T CACCGAC CAGAAAGGCAATA
CCATCGTCGGCTCTGGCAGCGGCGGCACCACCAAGTACT T TAGAATCCCCGCCAT GT GCACCAC
CAGCAAGGGCACCAT T GT GG T GT T CGCCGACGCCAGACACAACACCGC CAGCGAT CAGAGCT IC
A_TCGATACCGCTGCCGCCAGAAGTACAGACGGCGGCAAGACCTGGAACAAGAAGATCGCCATCT
ACAAC GACCGCGT GAACAGCAAGC TGAGCAGAGT GAT GGACCC TACC T GCAT CGT GGC CAACAT
C CAGGGCAGAGAAACCAT CC T GGT CAT GGT CGGAAAGT GGAACAACAAC GATAAGACC T GGGGC
GCC TACAGAGACAAGGCCCC T GATACCGAT T GGGACC T CG T GC T GTATAAGAGCACCGAC GAC G
GCGTGACCTTCAGCAAGGTGCAAACAAACATCCACGACATCGTGACCAAGAACGGCACCATCTC
T GCCATGC T CGGCGGCGT T GGAT C TGGCC T GCAAC T GAAT GAT GGCAAGC T GGT GT T
CCCCGT G
CAGAT GGT CCGAACAAAGAACAT CAC CACCGT GC T GAATAC CAGCT T CAT C TACT CCACCGAC G
GCAT CACAT GG T CCCT GCC TAGCGGC TAC T GT GAAGGCT T TGGCAGCGAGAACAACATCATCGA
GT T CAACGC CAGC C T GG T CAACAACAT C C GGAACAGC GG C C T GC GGAGAAGC T
TCGAGACAAAG
GAC T TCGGAAAGACGT GGACCGAGT T ICC TCCAAIGGACAAGAAGGT GCACAACCGGAAC CAC G
GCGTGCAGGGCAGCACAATCACAATCCCTAGCGGCAACAAACTGGTGGCCGCTCACTCTAGCGC
C CAGAACAAGAACAAC GAT TACAC CAGAAGCGACAT CAGCC T GTACGCCCACAACC T G TACT CC
GGCGAAGTGAAGCTGATCGACGACTTCTACCCCAAAGIGGGCAATGCCAGCGGAGCCGGCTACA
GC T GT C TGAGC TACCGGAAAAAT GTGGACAAAGAAACCC T GTACGTGGT GTACGAGGCCAACGG
CAGCATCGAGT T T CAGGACC T GAGCAGACAT C T GCCCGT GAT CAAGAGC TACAAT GGCGGAGGT
GGAAGTGGCGGAGGCGGAT C CGACAAAAC T CACACATGC CCACCGTGC C CAGCACC T GAACT CC
T GGGGGGACCG T CAGT C T T CC TCT TCCCC CCAAAACCCAAGGACACCC T CAT GAT C T CC
CGGAC
C CC T GAGGT CACATGCGT GG T GGT GGACG T GAGCCACGAAGACCCTGAGGT CAAGT T CAACT GG
TACGT GGACGGCGTGGAGGT GCATAAT GC CAAGACAAAGCCGC GGGAGGAGCAGTACAACAGCA
C GTACCGT GT GGT CAGCGT CC TCACCGT CC T GCACCAGGAC T GGCTGAATGGCAAGGAGTACAA
G T GCAAGGT C T CCAACAAAGCCCT CCCAGCCCCCAT CGAGAAAACCAT C T CCAAAGCCAAAGGG
CAGCCCCGAGAACCACAGGTCTAcAccrTGcccccATCCCGGGAGGAGATGACCAAGAACCAGG
T CAGCC TGAC C T GCCT GGT CAAAGGC T IC TAT CCCAGCGACAT CGCCGT GGAGT GGGAGAGCAA
T GGGCAGCCGGAGAACAAC TACAAGACCACGCC T CCCGT GC T GGACT CC GACGGC T CC T T CT
IC
C T CAC TAGCAAGC TCACCGT GGACAAGAGCAGGTGGCAGCAGGGGAACG T C T TCT CAT GC TCCG
T GAT GCAT GAGGC TCT GCACAACCAC TACACGCAGAAGAGCC T C TCCC T GT C TCCGGG TAAAAG
C GGCGGAGGC GGATCT CAT CATCACCAT CAT CACCATCAC
105861 SEQ ID NO: 108:
TVEKSVVFK_AEGEHFTDQKGNT IVGS GS GGT TKYFRI PAMC T T SKGT IVVFADARHNTASDQS F
I DTAAARS TDGGKIWNKKIAIYNDRVNSKLSRVMDPTCIVANIQGRET I LVMVGKWNNNDKTWG
AYRDKAPDTDWDLVLYKS TDDGVT FSKVE TNI HD IVTKNG T I SAMLGGVGS CLQLNDCKLVFPV
QMVRTKNITTVLNTSFIYS TDGITWSLPSGYCEGFGSENNI IEFNASLVNNIRNSGLRRSFETK
D FGKTW TE FP PMDKKVDNRNHGVQGS TI TIPS GNKLVAAHS SAQNKNNDYTRSDI SLYAHNLYS
GEVKL I DD FY PKVGNAS GAGY S CL SYRKNVDKE T LYVVYEANGS I E FQDL S RHLPVI KS
YNGGG
GS GGGGSDKT HT CPPCPAPE LLGGPSVFL FPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYGS TYRVVSVL TVLHQDWLNGKE YKCKVS NKAL PAP I EKT I SKAKG
QPRE PQVYT L P P SREEMTKNQVS L TCLVKGFYP S D IAVEWE SNGQPENNYKT TPPVLDS DGS FF
L TSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGKSGGGGSHHHHHHHH
166
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105871 SEQ ID NO: 109:
ACAGT GGAAAAGT CCGTGGT GTT CAAGGCCGAGGGCGAG CAC T TCACCGACCAGAAAGGCAATA
C CAT CGTCGGC TO TGGCAGC GGCGGCACCACCAAGTACT T TAGAATCCC CGCCAT GT GCACCAC
CAGCAAGGGCACCAT T GT GG T GT T CGCCGACGCCAGACACAACACCGC CAGCGAT CAGAGCT T C
AT CGATACCGC T GCCGC CAGAAGTACAGAC GGC GGCAAGACC T GGAACAAGAAGAT CGCCAT C T
A_CAAC GACCGCGT GAACAGCAAGC TGAGCAGAGT GAT GGACCC TACC T GCAT CGT GGC CAACA T
C CAGGGCAGAGAAACCA.T CC T GGTCAT GGICGGAAA.GT GGAACAACAAC GATAA.GA.CC T GGGGC
GCCTA.CAGAGACAAGGCCCCTG.ATA.CCGAT T GGGA.CC T CG T GC T GTAT.AAG.AGCA.CCG.AC
GAC G
GCGTGACCT TCAGCAAGGTGGAAACAAACATCCACGACATCGTGACCAAGAACGGCACCATCTC
TGCCATGCTCGGCGGCGT T GGAT C TGGCC T GCAAC T GAAT GAT GGCAAGC T GGT GT T CC
CCGT G
CAGAT GGT CCGAACAAAGAACAT CAC CAC C G T GC T GAATACCAGCT T CAT C TACT C CAC C
GAC G
GOAT C.ACAT GG T CCCT GCC TAGCGGC TAC T GT GAAGGCT T TGGCAGCGAGAACAACATCATCGA.
GT T CAACGCCAGCCTGGT CAACAACAT CCGGAACAGCGGCC T GC GGAGAAGC T T CGAGACAAAG
GACT TCGGAAAGACGTGGACCGAGTT T CC T CCAAT GGACAAGAAGGT GGACAACCGGAACCAC G
GCGTGCAGGGCAGC.AC.AATCACAATCCCTAGCGGCAACAAACTGGIGGCCGCTCACTCTAGCGC
C CAGAACAAGAACAAC GAT TACAC CAGAAGCGACAT CAGCC T GTACGCCCACAACC T G TACT CC
GGCGAAGTGAAGCTGATCGACGACTTCTACCCCAAAGIGGGCAATGCCAGCGGAGCCGGCTACA
GC T GT C TGA.GC TACCGGAAAAAT GTGGACAAAGAAACCC T GTACGTGGT GTACGAGGC CAACGG
CAGCATCGAGT T T CA.GGACC T GAGCAGACAT CT GCCCGT GAT CAAGA.GC TACAA.T GGCGGAGGT
GGAAGTGGCGGAGGCGGAT C CGACAAAAC T CACACATGC CCACCGTGC C CAGCACC T GAACT CC
T GGGGGGACCG T CAGT C T T CC TCT TCCCC CCAAAACCCAAGGACACCC T CAT GAT C T CC
CGGAC
C CC T GAGGT CACATGCGT GG T GGT GGACG T GAGCCACGAAGACCCTGAGGT CAAGT TCAACTGG
TACGT GGACGGCGTGGAGGT GCAT.AAT GC CAAGACAAAGCCGC GGGAGGAGCAGTACGG TAGCA
CGTACCGT GT GGT CAGCGT CC TCACCGT CC T GCACCAGGAC T GGCTGAAT GGCAAGGAG TACAA
GTGCAAGGTCTCC.AACAAA.GCCCTCCCA.GCCCCCA.TCGAG.AAAA.CCATCTCCAAA.GCCAAAGGG
CAGCCCMAGAACCACAGGTCTACACCOTC1CCCCCATOCCGCrfAC;C:rAGATGACCAAGAACCAGG
TCAGCCTGA.CCTGCCIGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA
T GGGCAGCCGGAGAACAAC TACAAGACCACGCC T CCCGT GC T GGACT CC GACGGC T CC T T CT
IC
C T CA.0 TAGC.AAGC TCACCGT GGAC.AAGAGCAGGIGGCAGCAGGGG.AACG T C T TCT CAT GC
TCCG
T GAT GCAT GAGGC TCT GCACAACCAC TACACGCAGAAGAGCC T C TC.CC T GT C TCCGGG
TAAAAG
C GGCGGAGGC GGATCT CAT CATCACCAT CAT CACCATCAC
105881 SEQ ID NO: 110:
EVQLLESGGGLVQPGGSLRLSC.AASGET FS S Y IMMWVRQA.PGKGLEWVS S I YPS GG I T FYADTV
KGRFT I SRDNSKNTLYLQMNS LRAEDTAVYYCARI KLGTVT TVDYWGQG T LVTVS SAS TKGPSV
FPLAP S SKS T S GGTAALGCLVKDY FPE PVTVSWNS GALT SGVHT FPAVL QS S GLYS L S
SVVTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
E YKCKVSNKAL PAP I EKT I SKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEW
E SNGQPENNYKT TPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GKGGGGSGGGGSGGGGSQSAL TQPA.SVS GS PGQS I T ISCT GT S S DVGGYNYVSWYQQHP GKAPK
LM I YDVSNRP S GVSNRFS GS KSGNTAS LT I S GLQAEDEADYYCS SYT S S S TRVFGT GT
KVTVLG
QPKAGGGGS GGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFT FS SYIMMWVRQAPGKGL
EWVSS IYPS GG I T FYADTVKGRFT I SRDNSKNT LYLQMNS LRAEDTAVYYCARI KLGTVT TVDY
WGQGTLVTVS S
167
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105891 SEQ ID NO: 111:
GAGGT GCAGC T GC TGGAAT C T GGCGGAGGA.0 T TGT TCAGCC T GGCGGC TCTC TGA.GAC T
GTC T T
G T GCCGCCAGCGGCT T CACC T TCAGCAGC TATAT CATGAT GT GGGTCCGACAGGCCCC T GGCAA
AGGCCITGAATGGGIGTCCAGCATCTATCCCAGCGGCGGCATC.ACCT T T TACGCCGACACAGTG
AAGGGCAGAT T CAC CAT CAG C CGGGACAACAG CAAGAACAC CCTG TAC C T GCAGAT GAACAGCC
TGAGAGCCGA_GGACACCGCCGTGTACTACTGCGCCAGAATCAAGCTGGGCACCGTGACCACCGT
GGAT TAT T GGGGAC.AGGGCACCCIGGICACCGT =AT C T GC T.AGCACCAAGGGCCCAT CCGT C
T T CCCCCTGGCACCCT CC T CCAAGA.GCA.CCTC T GGGGGCA.CAGCGGCCC T GCGCT GCC T
GGTCA.
AGGACTACT T CCCCGAACCGG TGACGGT G T CC T GGAACT CAGGCGCT C T GACCAGCGGCGTGCA
CACCT TCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
TCCAGCAGCT TGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG
T GGACA_AGAAAGT TGAGCCCAAAT CT T GT G.AC.AAAAC T CA.CACAT GC C CAC C GT GC C
CAG CAC C
T GAAC TCC T GGGGGGA.CCGT CAGT CT T CC TCT T CCCCCCAAAACCCAAGGACACCC T CAT GAT
C
TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
T CAAC TGGTAC GT GGACGGC G TGGAGGT GCATAAT GC CAAGACAAAGC C GC GGGAGGAG CAG
TA.
CAACAGCACG TACCGT GT GG T CAGCGT CC T CACCGT CCT GCACCAGGA_C T GGCT GAAT
GGCAAG
GAG TACAAG T G CAAGG ICT C CAAC.AAAGC CC T CC CAGCC C C CAT CGAGAAAAC CAT C T
CCAAAG
C CAAAGGGCAGCCCCGAGAACCACAGGT C TACACCC T GC C C C C.AT CC C GGGAGGAGAT
GACC.AA
GAACC.AGGT CAGCCIGTAC T GCCT GGTCAAAGGC T TC TAT CCC.AGCGACAT CGCCGT GGAGTGG
GAGAGCAAT GGGCAGCCGGAGAACAAC TACAAGAC CACGCC T CCCGT GC T GGACT CCGAC GGC T
CC T TC T TCC T C TATAGCAA.GC TCACCGT GGACAAGAGCAGGT GGCAGCAGGGGAACGT C T TCTC
AT GC T CCGT GAT GCAT GAGGC TCT GCACAACCAC TACAC GCAGAAGAGCC TAAGC TTGTC TCCG
GGTAAAGGAGGCGGAGGAT C T GGCGGAGG T GGAAGT GGC GGAGGCGGAT CT CAT CT GC T CT TA
CACAGCCT GCCAGCGT GT CCGGAT CT CCTGGCCAGAGCAT CACCATCAGC T GTACCGGCACCAG
CTCT GATGT CGGCGGC TACAAT TA.= GICC T CGT.ATCAGCAGCACCCCGGCAAGGCCCC TAAG
CTGATGATCTACG'ACGTGTCCAACAGACCCAGCGGCGTGTCCAATAGATTCTCCGGCAGCAAGA
GCGGCAACACCGCCAGCCTGACAATTAGCGGACTGCAGGCCGAGGACGAGGCCGAT TACTACTG
TAGCAGCTACACCAGC T CCAGCACCAGAG T GT T T GGCAC CGGCACAAAAGT GACCGT GC T GGGC
CAGCCT.AAGGCCGGIGGA.GGTGGGICTGGA.GGGGGIGGATCTGGAGGTGGCGGATCGGAGGIGC
AGC T GC TGGAAT C TGGCGGAGGAC T T GT T CAGCCT GGCGGC T C T CTGAGAC T GTC TTGT
GCCGC
CAGCGGCT T CACC T TCAGCAGCTATAT CAT GAT GT GGGT CCGACAGGCC CC T GGC.AAAGGCC T
T
GAAT GGGT GT C CAGCAT C TAT CCCAGCGGCGGCAT CACC T TI TACGCCGACACAGTGAAGGGCA
G_AT T C AC CA T CAGCCGGGACAACAGCAAGAACAC C C TGTA C C T GCAGA_T GAACAGCC T
GAGAGC
CGAGGACACCGCCGTGTAC TACT GCGCCAGAAT CAAGCT GGGCACCGT GACCACCGT GGAT TAT
T GGCGACAGGGCACCCICGT CACCGT GT CAT CT
105901 SEQ ID NO: 112:
DAS L PYLQKE SVFQSGAHAYR PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDEQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTD.AAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SA.FC FL SHDHGRTWARGH EVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVEPPPQGCQGSVI S FP S PRS GPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQGGGG
S GGGGSDKTH T CP PCPAPE L LGGP SVFL FP PKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQ
PRE PQVYT L P P SREEMIKNQVSLICLVKG FYP S D IAVEWE SNGQPENNYKT TPPVLDSDGSFFL
T SKL TVDKS RWQQGNVFS CSVMHEALHNHYT QKS L S L S P GK
168
CA 03173557 2022- 9- 27
WO 2022/150521
PCT/US2022/011504
105911 SEQ ID NO: 113:
GAT GCATC T C T GCCTTACC T GCAGAAAGAAAGCGT GIT CCAGT C TGGCGCCCACGCC TACAGAA
T T CCCGCT C T GC T GTAT C T GCCAGGCCAGCAGTC T C TGC T GGC T TTCGC T
GAACAGCGGGCCAG
CAAGAAGGAT GAGCAC GC C GAAC T GAT CG T GC T GC GGAGAGGC GAT TAC GAC GC C
GGCACACAT
CAGGT GCAGT GGCAGGC T CAAGAGGT GGT GGC T CAGGCTAGAC T GGAC GGCCACAGAT C TAT GA
A_CCCCTGICCTCTGTACGATGAACAGACCGGCACACTGT T TC T GTTC T T TAT CGC TAT CCCCGG
CCAAGTGACCGAGCAGCAGCAGCTGCAGACAAGAGCCAACGTGACCAGACTGIGICAAGTGACC
TCCACCGACCACGGCAGAACCTGGTCTAGCCCTAGAGATCTGACCGACGCCCCCATCGGACCTG
CC TATAGAGAG T GGTCCACC T TCGCCGT TGGACCTGGACAC T GT CTCCAGC T GCACGACAGGGC
TAGAT C TC T GG T GGTGCC T GCCTACGCC TATAGAAAGCT GCACCCCAAACAGCGGCC TAT TCC T
AGCGCC T TC T GC T TIC T GAGCCACGAT CACGGCAGGACAT GGGCCAGAGGACAT T T CG T
GGCCC
AG GACACAC T G GAAT GC CAG G TGGCC GAAGT GGAAAC CG GC GAG CAGAGAG T CGT GAC C
C T GAA
C GC CAGAT C T CAC C T GAGAG C CAGAG T GCAGGCCCAGAGCACAAACGACGGCC T GGAT T T
CCAA
GAGAGCCAGC T GGTCAAGAAACT GGT GGAACCT CC T CCACAGGGCTGT CAGGGAAGCG T GAT CA
GC T T T CCAT C T CC T.AGAAGCGGCCCT GGC T C T CC T GC T CAGT GGCTGC T
GTATACACACCCCAC
A_CACAGCT GGCAGAGAGCCGATCT GGGCGCC TACC T GAAT CC TAGACC T CC T GCT CC T GAGGC
T
T GGAGCGAAC C T GT TC T GOT GGCCAAGGGCAGCGC T GCC TACAGCGAT C T GCAGT C TAT
GGGCA
CAGGCCCT GAT GCCAGCCC T C TGT T T GGC T GIG T GTACGAGGCCAACGAC TACGAAGAGATCGT
G T T CC TGAT G T TCACCCTGAACCAGGCCITTCCAGCCGAGTACCTGCCTCAAGGCGGAGGTGGA
AGTGGCGGAGGCGGATCCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG
GGGGACCGT CAGT CT T CC T C T TCCCCCCAAAACCCAAGGACACCCTCAT GAT CT CCCGGACCCC
TGAGGIGACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTICAACTGGTAC
G T GGACGGCG T GGAGGT GCATAAT GCCAAGACAAAGCCGCGGGAGGAGCAG TACAACAG CACGT
ACCGT GTGGT CAGCGT CC T CACCGTCC T GCACCAGGACT GGC T GAAT GGCAAGGAGTACAAGT G
CAAGGTCT CCAACAAAGCCC T CCCAGCCCCCAT CGAGAAAAC CATCT CCAAAGCCAAAGGGCAG
CCCC.qA(21AACCACAGGTCTACACCCT=CCCATCCMqqAGqAqATGACCAAGAACCAGGTCA
GCC T GACC T GC C T GGT CAAAGGCT IC TAT CCCAGCGACAT CGCCGTGGAGT GGGAGAGCAAT GG
GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT T CC T C
AC TAGCAAGC T CACCGIGGACAAGAGCAGGIGGCACCAGGGGAACGT C T TC T CAT GC T CCGT GA
TGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCIGTCTCCGGGTAAA
105921 SEQ ID NO: 114:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDEQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQS TNDGLDFQ
ESQLVKKLVEPPPQGCQCSVI S FP S PRS C PC S PAQWLLY THP THSWQRADLGAYLNPRP PAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQEPKS
S DKTHTCP PC PAPELLGGP SVFL FPPKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYNS TYRVVSVLTVLEIQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLT
VDKSRWQQGNVFS CSVMHEALHNHYT QKS L S L S PGKGGGGS GGGGSGGGGS QSAL T QPASVS GS
PGQS I T I SCT GT S SDVGGYNYVSWYQQHP GKAPKLMIYDVSNRP SGVSNRFS GSKS GNTASL T I
S GLQAEDEADYYCS S YT S S S TRVFGTGTKVTVLGQPKAGGGGSGGGGSGGGGSEVQLLESGGGL
VQPGGSLRLSCAASGFT FS SY IMMWVRQAPGKGLEWVSS I YP S GG I T FYADTVKGRFT I SRDNS
KNT LYLQMNS LRAEDTAVYYCARI KLGTVT TVDYWGQGT LVTVS S
169
CA 03173557 2022- 9- 27
WO 2022/150521
PCT/US2022/011504
105931 SEQ NO: 115:
GAT GC.ATC T C T GCCTTACC T GCAGAAA.GAAA.GCGT GIT CCAGT C TGGCGCCCACGCC
TACAGAA.
T T CCCGCT C T GC T GTAT C T GCCAGGCCAGCAGTC T C TGC T GGC T TTCGC T
GAA.CAGCGGGCCAG
CAA.GAA.GGAT GAGCAC GC C GAAC T GAT CG T GC T GC GGAGAGGC GAT TAC GAC GC C
GGCACACAT
CAGGT GCAGT GGCAGGC T CAA.GAGGT GGT GGC T CAGGCTAGAC T GGAC GGCCACAGAT C TAT
GA
A_CCCCIGICCTCTGIACGATGAACAGACCGGCACACIGITTCTGTICTTTATCGCTATCCCCGG
C CAAG T GAC C GAG C.AG CAG CAGC T GCAGACAAGAGCCAACGT G.ACCAGAC T G T GT CAAG
T GACC
TCCA.CCGACCA.CGGCA.GAA.CCIGGICTA.GCCCTA.G.AGATCTGACCGACGCCGCCA.TCGGACCTG
CC TATAGAGAG T GGTCCACC T TCGCCGT TGGACCTGGACAC T GT CTCCAGC T GCACGACAGGGC
TAGAT C TC T GG T GGTGCC T GCCTACGCC TATAGAAA.GCT GCACCCCAAA.CAGCGGCC TAT TCCT
AGCGCC T TC T GC T TTCTGAGCCACGATCACGGCAGGACATGGGCCAGAGGACATT TCGTGGCCC
AG GAC.ACAC T G GAAT GC CAG G T GGCC GAAG T GGAAAC CG GC GAG CAGAGAG T CGT GAC
C C TGAA.
C GC CAGAT C T CAC C T GAGAG C CAGAG T GCAGGCCCAGAGCACA_AACGAC GGCC T GGAT T T
CCAA.
GAGAGCCAGC T GGICAAGAAA.CT GGT GGAA.CC T CC T CCACAGGGCTGT CAGGGAA.GCG T GAT
CA.
GC T T T CCAT C T CC T.AG.AA.GCCGCCCT GGC T C T CC T GC T CAGT GGCTGC T
GTATACACACCCCAC
ACACAGCT GGCAGAGAGCCGATCT GGGCGCC TACC T GAA_T CC TAGACC T CC T GCT CC T GAGGC
T
T GGAGCGAA.0 C T GT TC I GOT GGCCAAGGGCAGCGC T GCC TACAGCGAT C T GCAGT C TAT
GGGCA
CAGGCCCT GAT GGCAGCCC T C TGT IT GGC I GTC T GTACGAGGCCAACGAC TACGAA.GAGATCGT
GT T CC TGAT G T T CACCC T G.A.AGCA.GGCCT T
TCCA.GCCGAGTACCTGCCTCAAGAGCCCAA_ATCT
TCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGICT
TCCTCTICCCCCCAAAA.CCCAAGG.ACACCCTCATGATCTCCCGGACCCCTGAGGICACATGCGT
GGTGGIGGACGTGAGCCACCAAGACCCTGAGGICAA.GTICAA.CTGGTACGTGGACGGCGTGGAG
G T GCATAAT GCCAAGACAA_AGCCGCGGGAGGAGCAG TACAACAGCACGTACCGT GT GG T CAGCG
T CC T CACC GT CC T GCACCAGGACT GGC T GAA.T GGCAA.GGAGTACAAGT G CAA.GGT C T
CCAACAA.
AGCCC T CC CAG C C CCCA.T C GAG.AA_AA.0 CAT C T CCA_AA.GC
CAAA.GGGCAGCCCCGA.GAAC CACA.G
GICTACACCCTGCCMCATCCCMGAGGAGATGACCAAGAACCAGC:fTCAGCCICrACCTGCCIGG
T CAAA.GGC T T C TAT CC CAGC GACAT C GC C GT GGAGT GGGAGAGCAA.T
GGGCAGCCGGAGAACAA.
C TACAA.GACCACGCCT CCCG T GCT GGAC T CCGACGGC T CC T TC T ICC T C TATAGCAAGC T
CACC
G T GG.A.C.AA.GAGC.AGGIGGCAGCAGGGG.AACGTC T TC TCAT GC T CCGT GA.T GCAT GAGGC
T CT GC
ACAAC CAC TACACGCAGAA_GAGCC TAAGC T T GT C T CC GG G TAAAGGAGGCGGAGGAT C T
GGCGG
AGGT GGAA.GT GGCGGAGGCGGAT C TCAA.T C T GC T C T TACACAGCCTGCCAGCGT GT CCGGAT
C T
CC T GGCCAGAGCATCACCAT CAGC TGTACCGGCACCAGC T C T GATGT CGGCGGCTACAA.T TACG
T GT CCIGGTAT CAGCAGCACCCCGGCAAGGCCCC TAAGC T GAT GATC TACGACGIGT CCA_ACAG
ACCCAGCGGCG T GICCAA.TAGAT T CT CCGGCAGCAA.GAGCGGCA_ACACCGCCAGCC T GACAAT T
AGCGG.ACT GCAGGCCGAGGAC GAGGC C GAT TAC TAC TGTAGCA.GCTACACCAGCT C CAG CAC CA
GAGT GI I I GGCACCGGCACAAAAGIGACC GI GC I GGGCCAGCC TAAGGC CGGIGGAGG T GGGIC
T GGAGGGGGT GGATCT GGAGG TGGCGGAT CGGAGGT GCAGC T GC TGGAA.T C T GGCGGAGGAC T
T
GT T CAGCC T GGCGGCT C TC T GAGACT GT C T T GT GCCGCCAGCGGCT T CACC T TCAGCAGC
TATA
T CAT G.ATGT GGGT CCGACAGGCCCCT GGCAA_AGGCCIT GAA.T GGGTGT CCAGCAT C TAT
CCCA.G
C GGC GGCAT CACC T TT TACGCCGACACAGTGAAGGGCAGAT TCACCATCAGCCGGGACA.ACAGC
AA.GAACACCCTGTACCIGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCG
CCAGAA.TCAAGCTGGGCA.CCGTGACCACCGTGGAT T.AT TGGGG.ACAGGGCACCCIGGTCACCGT
G T CAT CT
105941 SEQ ID NO: 116:
X LASLPX2LQX3E SVFQS GAHAYRI PALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAX4
T HQVQWQAQEVVAQARLDGHRSMNPCPLYDX5QT GT L FL FF 'AI P GQVTEQQQLQ T RANVTRLCX
6VTSTDHGRTWSSPRDL TDAAIGPAYREWS T FAVC-ITGHCLQLHDRARSLVVPAYAYRKLHPX7QR
P1 P SAFCFL S HDHGRTWARGH FVAQDT LECQVAEVE TGE QRVVT LNARS HLRX 8RVQAQS TNDGL
D FQE S QLVKKLVE PPPX 9GCQGSVI S FPS PRS GPGS PAQWLLYTHP THX10XliQRADLGAYLNPR
170
CA 03173557 2022- 9- 27
WO 2022/150521
PCT/US2022/011504
P PAPEAWSE PVLLAKGSX12AYS DLQSMGTGPDGS PL FGCLYEANDYEE IX i3FX14MFT LKQAFP
AEYLPQX1:DKTHT C PPCPAPELLGGPSVFL FPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREE QYNS TYRVVSVL TVLHQDWLNGKEYKCKVS NKAL PAP I EKT I SKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGS F
FL T SKL TVDKSRWQQGNVFS C SVMHEALHNHYTQKS LS L S PGK
105951 SEQ ID NO: 117:
X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEHAELIVX9RRGD
YDAXioTHQVQWX11AQEVVAQAX12LX13GHRSMNPCPLYDX14QTGTL FL FFIAI PX15X16VTEX17
QQLQTRANVTRLX18X19VTSTDHGRTWSSPRDLTDAAIGPX20YREWS T FAVGPGHX21LQLHDX22
X23RS LVVPAYAYRKLEPX24X25X26P I PSAFX27 FLSHDHGRTWARGHFVX28QDTX29ECQVAEVX
3uTGE QRVVTLNARSX3iX32X33X34RX3sQAQSX36NX37GLDFQX38X39QX4 oVKKL
X 41E P PPX42GX43QGSVI S FPS PRSGPGSPAQX44LLYTHPTHX45X16QRA_DLGAYLNPRPPAPEA
WSEPX47LLAKGSX48AYSDLQSMGTGPDGSPLFGX49LYEANDYEEIX50FX5iMFTLKQAFPAEYL
PQX52DKTPITCPPC PAPELLGGPSVELFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNS T YRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQPR
EPQVYTLPPSREEMTKNQVSLTGLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGS FELTS
KL TVDKSRWQQGNVFS CSVMHEALHNHYTQKS LS LS PGK
105961 SEQ ID NO: 118:
g a t GCATCTC T GCCTTACCT GCAGAAAGAAAGCGTGITCCAGTCTGGCGCCCACGCCTACAGAA
T TCCGGCTCTGCTGTATCTGCCAGGCCAGCAGICTGTGCTGGGTITCGCTGAACAGCGGGCCAG
CAAGAAGGAT GAGCAC GC C GAAC T GAT C G T GC T GC GGAGAGGC GAT TAC GAG GC C
ggcACACAT
CAGGT GCAGT GGCAGGGT CAAGAGGT GGT GGCT CAGGCTAGAC T GGAC GGCCACAGAT C TAT GA
ACCCGTGICCTCTGTAGGAT g aaCAGACCGGGAGAGIGT T TCTGT TGT T TATGGCTATCCCCGG
C CAAGTGACCGAGCAGGAGCAGCTGGAGAGAAGAGGCAACGTGACCAGACTGIGT t a cGTGACG
T CCACCGACCACGGCAGAACC TGGICTAGCCCTAGAGAT C TGACCGACGCCGCCATCGGACCTG
CCTATAGAGAGTGGICGACCTICGCCGTTGGACCTGGACACTGTCTCCAGCTGCAGGACAGGGC
TAGATCTCTGGTGGIGCCTGCCTACGGCTATAGAAAGCTGCACCCCAAACAGCGGCCTATTCCT
AGCGGGITCTGCTITCTGAGCCACGATGAGGGGAGGACATGGGGCAGAGGACATTTGGTGGCCG
AG GACACAC T G GAAT GC CAG G TGGCC GAAGT GGA_AAG CG GC GAG CAGAGAG T CGT GACCC
TGAA
CGCCAGATCTCACCTGAGATTCAGAGTGCAGGCCCAGAGCACAAACGACGGCC.TGGATTTCCAA
GAGAGGCAGCTGGICAAGAAACTGGTGGAACCTCCTCCAaccGGCTGTCAGGGAAGCGTGATCA
GCTTTCCATCTCCTAGAAGCGGCCCTGGCTCTCCTGCTCAGTGGCTGCTGTATACACACCCCAC
ACACAGCTGGCAGAGAGCCGATCTGGGCGCCTACCTGAATCCTAGACCTCCTGCTCCTGAGGCT
TGGAGGGAACCTGTICTGCTGGCCAAGGGCAGCgctGCCTAGAGCGATCTGGAGICTATGGGCA
CAGGCGCT GAT GGCAGCCCT C TGT IT GGC T GTCT GTACGAGGCCAACGAC TACGAAGAGATCGT
GT TCCTGATGT TCACCCTGAAGCAGGCCT T TCGAGGGGAGTAGGIGCC T CAA
105971 SEQ ID NO: 119:
X iASLPX2LQX3E SVFQS GAHAYRI PALLYLPGQQS LLAFAEQRASKKDEHAEL IVLRRGDYDAX4
THQVQWQAQEVVAQARLDGHRSMNPCPLYDX5QTGIL FL FFIAT PGQVTEQQQLQTRANVIRLCX
6VT S TDHGRTWS S PRDL T DAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPX7QR
P1 PSAFCFLS HDHGRTWARGH FVAQDTLECQVAEVE TGE QRVVTLNARS HLRX8RVQAQS TNDGL
D FQE S QLVKKLVE PPPX9GCQGSVI S FPS PRS GPGS PAQWLLYTHP THX10XliQRADLGAYLNPR
PPAPEAWSEPVLLAKGSX12AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX13FX14MFTLKQAFP
AEYLPQ
171
CA 03173557 2022- 9- 27
WO 2022/150521
PCT/US2022/011504
105981 SEQ ID NO: 120:
XiX2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEHAELIVX9RRGD
YDAXioTHQVQJXnAQEVVAQX2LX3GHRSMNPCPLYDXi4QTGTL FL FFIAI PX15X16VTEX17
QQLQTRANVTRLX10X19VTSTDHGRTWSSPRDLTDAA.IGPX20YREWS T FAVGPGHX21LQLHDX22
X23RS LVVPAYAYRKLPIPX24X25X26P I P SAFX27 FL SHDHGRTWARGH FVX28QDTX29ECQVAEVX
3oT GE QRVVTLNARSX31X32X33X34RX35QAQSX36NX37 GLDFQX38X39QX40VKKL
X41E P PPX42GX43QGSVI S FPS PRSGPGSPAQX44LLYTHPTHX45XI6QRADLGAYLNPRPPAPEA.
W SE PX47LLAKGSX48AYS DLQSMGT GPDGS PL FGX49LYEANDYEE I X50FX5iMFT
LKQ.AFPA.EYL
PQ
105991 SEQ ID NO: 121:
GGGGS
106001 SEQ ID NO: 122:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
OVOWQ.AQEVVAQARLDGHRSMNPCPLYDE QT GT L FL FFIAI PGOVTEQQQLOTRANVTRLCYVT
S TDHGRTWS S PRDLTDAA I GPAYREWSTFAVGPGHCLQLFIDRARSLVVPAYAYRKLHPKQRPI P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E S QLVKKLVE P PP TGCQGSVI S FP S PRS GPGSP.AQWLLYTHPTHSWQR.ADLGAYLNPRPPAPEA.
W SE PVLLAKGSA_AYSDLQSMGTGPDGS PL FGCLYEANDYEE IVELMFTLKOAFPAEYLPOEPKS
S DKTHTCPPC PAPELLGGP SVFL FPPKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKT ISKAKGQPIREPQ
VYT L PP SREEMTKNQVS LYCLVKGFYP S D IA.VEWE SNGQPENNYKT T P PVLDSDGS FFLYSKLT
VDKSRWQQGNVFS CSVMHEALHNHYTQKS L S LS PGK
106011 SEQ ID NO: 123:
DAS L PYLQKE SVFQSGA.HAYR I PALLYL P GQQS LLA.FAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQ.AQEVVAQARLDGHRSMNPCPLYDEQT GT L FL FFIA.I PGQVTEQQQLQTRANVTRLCYVT
S TDHGRTWS S PRDLTDAA.I GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLR FRVQAQS TNDGLDFQ
E S QLVKKLVE P PP TGCQGSVI S FP S PRS GPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA.
W SE PVLLAKGSAAYSDLQSMGTGPDGS PL FGCLYEANDYEE IVFLMFTLKQ.AFPAEYLPQEPKS
S DKTHTCPPC PAPELLGGP SVFL FPPKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
VYT L PP SREEMTKNQVS LYCLVKC FYP S D IAVEWE SNCQPENNYKT T P PVLDSDC S FFLYSKLT
VDKSRWQQGNVFS CSVMHEALHNHYTQKS L S LS PGK
106021 SEQ ID NO: 124:
EVQLLESGGGLVQPGGSLRLSCAA.SGET FS S Y IMMWVRQAPGKGLEWVS S I YPS GG I T FYADTV
KGRFT I SRDNSKNTLYLQMNS LRA.EDTAVYYCARIKLGTVT TVDYWGQGT LVTVS SAS TKGPSV
FPLAP S SKS T S GGTAALGCLVKDYFPE PVTVSWNS GALT SGVHT FPAVL QS S GLYS L S
SVVTVP
S S S LGTQTY I CNVNHKP SNTKVDKKVE PKS CDKTHT CPPC PAPELLGGP SVFL FPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALP.APIEKT I S KAKGQPRE PQVYT L PP SREEMTKNQVS L TCLVKGFYPSDIAVEW
F. SNGOPENNYKT T PPVLDS DGS FFLT
TVDKSRWOOGNVESCSVMHEALHNHYTOKSLST,S
GK
172
CA 03173557 2022- 9- 27
WO 2022/150521
PCT/US2022/011504
106031 SEQ NO: 125:
Q SAL T QPASVS GS PGQS IT IS CT GT S S DVGGYNYVSWYQQHPGKAPKLM I YDVSNRP S
GVSNRF
S GSKSGNTAS L T I SGLQAEDEADYYCS SYTSSS TRVFGTGTKVTVLGQPKAGGGGSGGGGSGGG
GSEVQLLES GGGLVQPGGS LRLSCAASGFT FS S Y IMMWVRQAPGKGLEWVS S IYPSGG I T FYAD
TVKGRFT I S RDNS KNT LYLQMNS LRAE DTAVYYCAR I KL G TVT TVDYWGQGTLVTVS S
106041 SEQ ID NO: 126:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDEQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCYVT
S TDHGRTWS S PRDLTDAAT GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQ
106051 SEQ ID NO: 127:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDE QT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCYVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQEPKS
S DKTHTCP PC PAPELLGGP SVFL FPPKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
VYTLPPSREEMTKNQVSLTCLVKGFYPS D IAVEWESNGQPENNYKTTPPVLDSDGS FFL TSKLT
VDKSRWQQGNVFS CSVMHEALHNHYT QKS L S LS PGK
106061 SEQ NO: 128:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDEQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCYVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
E SQLVKKLVE PPPQGCQGSVI S FP S PRS GPGS PAQWLLY THP THSWQRADLGAYLNPRP PAPER
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQGGGG
S GGGGSDKTH T CP PCPAPE L LGGP SVFL FP PKPKDT LMI SRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQ
PRE PQVYT L P P SREEMIKNQVSLICLVKG FYP S D IAVEWE SNGQPENNYKT TPPVLDS DGS FFL
T S KL TVDKS RWQQGNVFS CS VMHEALHNHYT QKS LSLSP GK
106071 SEQ ID NO: 129:
GHAFTSDS
106081 SEQ ID NO: 130:
I YPRS GNP
173
CA 03173557 2022- 9- 27
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PCT/US2022/011504
106091 SEQ ID NO: 131:
DYYGRYFDV
106101 SEQ ID NO: 132:
EVQLQESGAE LARPGASVKL S CKASGHAFT S DS INWVKQR I GQGLEW I GE I YPRS GNPYYNEKF
KGKATLTADKS SS TAYME LRS LT S EDSAVY FCAT DYYGRY FDVWGTGT TVTVSS
106111 SEQ NO: 133:
EDIYNR
106121 SEQ ID NO: 134:
GAT
106131 SEQ ID NO: 135:
QQYWS TPWT
106141 SEQ ID NO: 136:
D I QMTQSS FS FSVSLGDRVT I ICKASEDIYNRLAWYQQKPGNTPRLL I S GAT SLE TGVP SRFS G
S GS GKDYTL S I TS LQTEDVATYYCQQYWS TPWT FGGGTKLE IR
106151 SEQ ID NO: 137:
GYS FTDYY
106161 SEQ ID NO: 138:
I YPGSGNT
106171 SEQ NO: 139:
SYYYGSSYLFDY
106181 SEQ ID NO: 140:
EVQLQESGAELVRPGASVKLSCKASGYS FTDYY INWVKQRPGQGLEW IAR YPGS GNT YYNEKF
KGKATLTAEKS S ITAYMQISSLTSEDSAVYFCARSYYYGSSYLFDYWGQGTTLTVSS
106191 SEQ NO: 141:
QS I GT S
106201 SEQ ID NO: 142:
YAS
174
CA 03173557 2022- 9- 27
WO 2022/150521
PCT/US2022/011504
106211 SEQ NO: 143:
QQSNNWPFT
106221 SEQ ID NO: 144:
D I LL TQSPAI LSVSPGERVS FSCRAS QS I GT S I HWYQQRTNGS PRLL I KYASES I S GI
PSRFSG
S GS GTDFTLS INSVESEDI GDYYCQQSNNWP FT FGSGTKLE IK
106231 SEQ NO: 145:
GYT FTDYY
106241 SEQ NO: 146:
I NPNNGYT
106251 SEQ ID NO: 147:
SAAYYVLDD
106261 SEQ ID NO: 148:
EVQLQQSGPE LVKPGALVK I SCKA.SGYT FT DYYMNWVKKSHGRS LEW I GD INPNNGYTNYNQNF
KGKATLTVDKS SS TVYME LRS LT S E DSAVYYCARSAAYYVLDDWGQGT SVTVSS
106271 SEQ ID NO: 149:
KKVT I FGS I SV
106281 SEQ NO: 150:
NGA
106291 SEQ ID NO: 151:
LQNKEVPYT
106301 SEQ ID NO: 152:
DIVMTQSPASLAVSLGQKAT I SCKASKKVT I FGS I SVLHWYQQKPGQPPKL IYNGAKLESGVSA
RFSDSGSQNRS PFGNQLNFTLT I DPVEADDAATYYCLQNKEVPYT FGGGTELE IK
106311 SEQ NO: 153:
GDS I TSGY
106321 SEQ ID NO: 154:
I SYTGST
175
CA 03173557 2022- 9- 27
LZ -6 -ZZOZ LSSELT0
9L
Imdamxaon og
:991 :ON CFI OAS
1:1717901
CENSAS
:S9I :oNcjjOjs
[MO]
SSALL'ILLISCSMDEDXSODASE2IV3XXAVIGESLYIS SrIWIXVICES SJGIIIV\21(1C
,31dGILINIC[1\TV(IGI>15 I ME'150ad>30MAMHWAIGM 'NZ SS-V-1D S'ILLASV5dHATI
HVSSE0'10AE
017
:1791 :ONGil OAS
IMO]
X0'3DXSOOX0
: T :ON CII
OgS II17901
INCINTV(IG I
:Z9I :ON CR OS
[017901
OE
:191 :ON CII OAS
16901
NI E'1>II00D,3IMdA0AAOODAXAVUGHVHASS I I'LL G LL SOSDII2IGa Ct
AS SHULL SVLV1 Yr-Did SOScaMOOXMVTISNMONSSArLISOSSIDSTALLAMEDASA:VTIS S SO
Sinai G
:091 :ON OAS
[8901
IMdXDXXon OZ
:6CI :ON CR OgS
L901
SiarNI
CI
:8S1 :ON CR O'Is
19901
SNMONSSAIYISC
: LSI :oNaIOJs
ig901 OI
SSAIASISn5MXGTAlidn'IM550SV9XXIVICEIIASN'In'IXA01\DISIG2iII S I27123
SSIXS XOTAIX
MNMX5 I I SG5IASDYISqInSdIA'ISa5S 0'10/\2
:9c1 :OK CET OAS
it901
XONVOrIMDS0
:SSI :ON CFI
tOSITO/ZZOZSIVIDcl IZSOSI/ZZOZ OA%
WO 2022/150521
PCT/US2022/011504
106451 SEQ ID NO: 167:
S IVMTQTPKFLLVSAGDRVT I TCKASQSVSNDVIWYQQKPGQSPKLL I YYAS IRFTGVPDRFAG
SGYGTDFT FT INTVQAEDLAVYFCQQDYNSPWT FGGGTKLE IF
106461 SEQ NO: 168:
I DPANGNT
106471 SEQ NO: 169:
P FNYRFYDVYYFDY
106481 SEQ ID NO: 170:
EVQLQESGAE LVKPGASVKL S CTASGFNIKDTYMHWVKQRPEQGLEW I GR DPANGNTKYDPKF
PGKAT ITADTS SNTAYLQLS S LTSEDAAVYYCARP FNYRFYDVYYFDYWGQGT TL TVS T
106491 SEQ NO: 171:
S SVSY
106501 SEQ ID NO: 172:
DT S
106511 SEQ ID NO: 173:
QQWS TYPLT
106521 SEQ ID NO: 174:
Q IVL TQS PAIMSAS PGEKVTMTCSAS S SVSYMYWYQQKPGS S PRLL I YDT SNLAS GVPLRFS GS
GS GT SYSL TL SRMEAEDAAT YYCQQWS TYPL T FGAGTKLELK
106531 SEQ ID NO: 175:
GYT FT S YV
106541 SEQ ID NO: 176:
INPYNDGS
106551 SEQ ID NO: 177:
QTLDF
106561 SEQ ID NO: 178:
EVQLQESGPELVKPGTSVKMSCKASGYT FT SYVMHWVKQRPGQGLEW I GY INPYNDGSKYNEKF
KGKATL TS DT S SS TAYMELS SLTSEDSAVYYCAKQTLDFWGQGTSVTVS T
177
CA 03173557 2022- 9- 27
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[0657] SEQ ID NO: 179:
ESVEFYGTTL
[0658] SEQ NO: 180:
AAS
[0659] SEQ NO: 181:
QQSRKVPYT
[0660] SEQ ID NO: 182:
D IVL TQSPA.S LAVSLGQRA.T I SCRASESVEFYGTTLMQWYQQKPGQPPKLL IYAASNVESGVPA
RFSGSGSGTDFSLNIHPVEEGDIGMYFCQQSRKVPYTFGGGTKLEIK
[0661] SEQ NO: 183:
GFSLS TYGLG
[0662] SEQ ID NO: 184:
I WWNDDK
[0663] SEQ ID NO: 185:
T LHYYDG TAW FAY
[0664] SEQ NO: 186:
QVTLKESGPG I LQPSQTLS L TCS FSGFS L S TYGLGVGWIRQPSGKGLEWL.ANIWWNDDKFYDSV
LKSRL T I SKDT SNNQVFLKI S SVDTSETATYYCAQTLHYYDGIAWFAYWGQGTLVTVSA
[0665] SEQ NO: 187:
HYVGT F
106661 SEQ NO: 188:
STS
[0667] SEQ NO: 189:
QQYYNSPLT
[0668] SEQ ID NO: 190:
DIVMTQSQNFMS TSVGDRVSVICKASHYVGT FVAWYQQKPGQSPKAL I FS TSYRHTGVPDRFTG
S GSGTDFTL T I SNVQSEDLADYFCQQYYNSPLT FGAGTKLELK
178
CA 03173557 2022- 9- 27
WO 2022/150521
PCT/US2022/011504
[0669] SEQ ID NO: 191:
GYT FT SNW
[0670] SEQ ID NO: 192:
I HPSDSET
[0671] SEQ NO: 193:
S SGDYGRDY
[0672] SEQ ID NO: 194:
QVQLQQPGAELVKPGASVKLSCKASGYT FT SNWMNWVKQRPGRGLEW I GRIHPSDSETHYHQKF
KSKATLTVDKS SS TAY I QLS SLTSEDSAVYYCAHSSGDYGRDYWGQGT T L TVS S
[0673] SEQ NO: 195:
ESVDSYGNS F
[0674] SEQ ID NO: 196:
LAS
[0675] SEQ ID NO: 197:
QQNNEDPWT
[0676] SEQ NO: 198:
NIVLTQSPASLAVSLGQRAT I SCRASESVDSYGNS FMHWYQQKPGQPPKLL IYLASNLQSGVPA
RFS GS GSRTDFTL T IDPVEADDAATYYCQQNNEDPWTFGGGTKLEIK
[0677] SEQ ID NO: 199:
EVQLVQS GAEVKKPGATVK I S CKVS G FN I KDT YMHWVQQAP GKGLEWMGL I DPANDNT I YAEKF
QGRVT I TADT S TDTAYMELS SLRSEDTAVYYCAREGYGGSYGEGYWGQGTLVTVSS
[0678] SEQ ID NO: 200:
E IVMTQSPPTLSLSPGERVTLSCRASQSVSNDLSWYQQKPGQAPRLL I YYAS IRFTGI PARFSG
S GS GTDFTL T I SSLQPEDFAVYYCQQDYNSPWT FGQGTKVE IK
[0679] SEQ ID NO: 201:
E IVMTQSPPTLSLSPGERVTLSCRASQSVSNDLSWYQQKPGQAPRLL I YYAS IRFTGI PARFSG
S GYGTDFTL T I SSLQPEDFAVYYCQQDYNSPWT FGQGTKVE 1K
179
CA 03173557 2022- 9- 27
WO 2022/150521
PCT/US2022/011504
106801 SEQ ID NO: 202:
E IVMTQSPPTLSLSPGERVTLSCRASQSVSNDLSWYQQKPGQAPRLL I YYAS IRFTGI PARES G
S GS GTDFTL T I SSLQPEDFAVYFCQQDYNSPWT FGQGTKVE IK
106811 SEQ ID NO: 203:
QQDYTSPWT
106821 SEQ ID NO: 204:
E IVMTQSPPTLSLSPGERVTLSCRASQSVSNDLSWYQQKPGQAPRLL I YYAS IRFTGI PARFSG
S GS GTDFTL T I SSLQPEDFAVYYCQQDYT SPWT FGQGTKVE IK
106831 SEQ ID NO: 205:
E IVMTQSPPTLSLSPGERVTLSCRASQSVSNDLSWYQQKPGQAPRLL YYAS IRFTGI P_ARFSG
S GS GTDFTL T I SSLQPEDFAVYYCQQDYTSPWT FGQGTKVE IKRTVAAP SVFI FPPS DE QLKS G
TASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLS S TLTLSK_ADYEKHKVY
ACEVTHQGLS S PVTKS FNRGEC
106841 SEQ ID NO: 206:
EVQLVQS CAEVKKPGATVK I S CKVS G FN I KDTYMHWVQQAPCKGLEWMGL I DPANDNT I YAEKF
QGRVT ITADTS TDTAYMELS S LRSEDTAVYYCAREGYGGSYGEGYWGQGTLVTVS SAS TKGPSV
FPLAPSSKS T S GGTAALGCLVKDYFPEPVTVSWNS GALT SGVHT FPAVLQSSGLYSLS SVVTVP
S S SLGTQTY I CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
S RT PEVTCVVVDVSHE DPEVK FNWYVDGVEVHNAKTKPREE QYAS TYRVVSVLTVLHQDWLNGK
EYKCKVSNKAL PAP IEKT I SKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GK
106851 SEQ ID NO: 207:
DASLPYLQKE SVFQSGAHAYR I PALLYLPGQQSLLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQVIJQAQEVVAQARLDGI-IRSIVINPCPLYDEQTGTLFLFFIAI PGQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SA FC FL SHDHGRTWARGH FV_AQDT LE CQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
ESQLVKKLVEPPPQGCQGSVI SFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
WSEPVLLAKGSAAYSDLQSMGTGPDGSPL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQEPKS
SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYAS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FEL T SKL T
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
106861 SEQ ID NO: 208:
GAAATTGTGATGACACAGAGCCCTCCAACGCTGAGCCIGTCTCCIGGCGAAAGAGTGACCCTGA
GC T GTAGAGC CAGCCAGAGC G TGT CCAAC GACC T GAGCT GGTAT CAGCAGAAGCC T GGACAGGC
CCCTCGGCTGCTGATCTACTACGCCAGCATCAGATTCACAGGCATCCCCGCCAGATTT TCCGGC
A_GCGGCTCTGGCACAGATT TCACCCTGACCATAAGCAGCCTGCAGCCTGAGGACTTCGCCGTGT
AC TAC T GT CAGCAGGAC TAC a ctAGCCCC TGGACCT T T GGCCAGGGCAC CAAGGT GGAAATCAA
180
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GCGTACGGT GGC T GCACCAT C TGT CT T CATCT T CCCGCCATCT GATGAGCAGT T GAAAT C
TGGA
ACTGCCTCTGT TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA.CAGTGGAAGG
TGGA.TAACGCCCTCCAATCGGGT.AACTCCCAGGAG.AGIGTCAC.AG.AGCAGGACA.GCAAGG.ACA.G
CA_C C TACAGCC T CAG'CA_GCA_CCCT G'AC G. C T G'AG'CAAAG'CA_GAC
TACGA_G'AAACACAAA_G. T C TA_C
GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAA.GAGCT TCAACA.GGGGAGAGT
GT
106871 SEQ ID NO: 209:
GAGGTGCA.GC T GGT TCAGT C TGGCGCCGAAGTGAAAAAGCCTGGCGCCACCGTGAAGAT CAGCT
GCAAGGIGTCCGGCTICAACATCAAGGACACCTACATGCACTGGGIGCAGCAGGCCCCTGGCAA
AGGACT TGAAT GGATGGGCC TGATCGACCCCGCCAAC GACAATACCAT C TAC GCCGAGAAGT IC
CAGGGC.AGAGT G.ACCAT CAC C GC C GACAC C TC TAC C GACAC C GC C TACAT GG.AAC T
GAG C.AGC C
T GAGAAGCGAGGACACCGCC G TGTAC TAC T GT GCCAGAGAAGGC TACGGCGGCA.GC TAC GGCGA
AGGATAT TGGGGAC.AGGGCACCCIGGICACCGT TAGCTC TGCt a gcACCAAGGGCCCAT CcGTC
T TCCCCCIGGCACCCTCCTCCAAGA.GCACCICTGGGGGCACAGCGGCCCTGGGCTGCCTGGICA.
A_GGACTACTTCCCCGAACCGGTGACGGTGICcTGGAACTCAGGCGCtCTGACCAGCGGCGTGCA
CACCT TCCCGGCT GTCC TACAGT CCT CAGGAC IC TAC IC CCT CAGCAGC CT GGT GACCGT GCCC
TCCACCAGCT TGGGCACCCAGACCTACATCTGCAACGTGAATC.AC.AAGCCCAGCAACACCAAGG
T GGACAAGAAAGT TGA.GCCCAAATCT TGTGA.C.AAAA.CTCACAC.ATGCCCACCGTGCCCAGCA.0 C
T GAAC TCCT GGGGGGACCGT CAGT CT T CC TCT T CCCCCCAAAACCCAAGGACACCCT CAT GATC
TCCCGGACCCCTGAGGICACATGCGTGGIGGIGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
T CAAC TGGTAC GT GGACGGC G TGG.AGGT GCATAAT GCCAAGACAAAGC C GCGGGAGGAGCAGTA
CgccAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG
GAG TACAAGT GCAAGGTCT CCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCT CCAAAG
CCAAA.GGGCAGCCCCGA.GAACCACA.GGTcTA.CA.CCCTGCCCCCATCCCGGGAGGA.GATGACCAA
GAACCAGGITCAGCCTG'ta cTGCCTGGTCAAAGC_Ir'CTTCTATCCCAGCGACATCCIrCILITGGACIr'TGq
GAGAGCAATGGGCAGCCGGAGAACAAC TACAAGAC CACGCCTCCCGT GC TGGACTCCGAC GGC T
CCT TCTTCCT C TATAGCAAGC TCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGT C T TCTC
ATGCTCCGTGATCCATGAGGCTCTGCAC.AACCACTACACGCAGAAGA.GCCTAAGcTTGTCTCCG
GGTAAA
106881 SEQ ID NO: 210:
g a t GCATCTC T GCCTTACCT GCAG.AAAGAAAGCGTGT TCCAGTCTGGCGCCCACGCCTACAG.AA
T TCCCGCTCTGCTGTATCTGCCAGGCCAGCAGICTCTGCTGGCTITCGCTG.AAC.ACCGGGCCAG
CAAGAAGGAT GAGCAC GC C GAAC T GAT C G T GC T GC GGAGAGGC GAT TAC GAC GC C g g
cACACAT
CAGGTGCAGTGGCAGGCTCAAGAGGTGGTGGCTCAGGCTAGACTGGACGGCCACAGATCTATGA
ACCCCTGICCTCTGTACCATgaaCAGACCGGCACA.CTGITTCTGITCTTTATCGCTA.TCCCCGG
CCAAGTGACCGAGCAGCAGCAGCTGCAGACAAGAGCCAACGTGACCAGACTGIGICAACTGACC
TCCACCGACCACGGCAGAA.CCIGGICTAGCCCTAGAGATCTGACCGACGCCGCCATCGGACCTG
CCTATA.GAGAGTGGICCACCTICGCCGTIGGACCIGGA.CACTCTCTCCAGCTGCACGACAGGGC
TAGATCTCTGGTGGIGCCTGCCTA.CGCCT.ATAGAAAGCTGCACCCCAAACAGCGGCCTATTCCT
AGCGCCTTCTGCTTTCTGAGCCACGATCACGGCAGGACATGGGCCAGAGGACATTTCGTGGCCC
AGGACACACTGGAATGCCAGGTGGCCGAAGTGGAAACCGGCGAGCAGAGAGTCGTGACCCTGAA
CGCCAG.ATCTCACCTGAGAGCCAGAGTGCAGGCCCAGAGCACAAACGACGGCCIGGAT T TCCAA
GAGAGCCAGCTGGICAAGAAACTGGTGGAACCTCCTCCACAGGGCTGTCAGGGAAGCGTGATCA
GCTTTCCATCTCCTAGAAGCGGCCCTGGCTCTCCTGCTCAGTGGCTGCTGTATACACACCCCAC
ACACAGCTGGCAG.AGA.GCCGATCTGGGCGCCTA.CCTG.AATCCTAG.ACCTCCTGCTCCTGAGGCT
T GGAGCGAACC TGTTC TGCT GG'CCAAGGGCACCgc t GCC TACACCGAT C TGCAGTC, TAT GGGCA
CAGGCCCTGATGGCAGCCCTCTGITTGGCTGICTGTACGAGGCCAACGACTACGAAGAGATCGT
GT TCCTGATGT TCACCCTGAAGCAGGCCT T TCCAGCCGAGTACCTGCC T CAAGAGCCCAAATCT
181
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T cTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCT
T CC T C T TCCCCCCAAAACCCAAGGACACCC T CAT GATCT CCCGGACCCC T GAGGT CACAT GCGT
GGT GGIGGACGT G.AGCCACGAAGA.CCC T G.AGGIC.AAGT C.AA.0 T GGTACGT GGA.CGGCGT
GGA.G
G T GCATAAT GC CAAGACAAA_GCCG'CGGGAGGAGCA_GTA_C g c cAGCA_CGTACCGT GT GG T
CAGCG
T CC T CACCGT CC T GCAC CAG GACT GGC T GAAT GGCAAGGAG TACAAGT GCAA.GGIC T
CCAAC.AA
AGCCCICCCAGCCCCCATCGAGAAAACCAICICCAAAGCCAAAGGGCAGCCCCGAGAACCACAG
GT c T ACACCC T GCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGT CAGCC TGACC T GCCTGG
T CAAA.GGC T T C TAT CC CAGC GACA.T C GC C G T GGAGT GGGAGAGCAAT
GGGCAGCCGGAGAACAA.
CTACAAGACCACGCCTCCCGTGCTGGA.CTCCGACGGCTCCTICTICCTCacc.AGC.AAGCTCA.CC
GT GGACAAGAGCAGGIGGCAGCAGGGGAACGIC TC TCAT GC T CCGT GAT GCAT GAGGC T CT GC
ACAAC CAC TACAC GCAGAA.GAGCCICICCCIGT CTCCGGG TAAA
[0689] SEQ ID NO: 211:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQA.QEVVAQARLDGHRSMNPCPLYDEQIGIL FL FFIAI PGQVTEQQQLQTRANVTRLCYVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRT WARGH FVAQDT LE CQVAEVE T GE QRVVT LN.ARS HLR FRVQAQS TNDGLDFQ
E S QLVKKLVE P PP TGCQGSVI S FP S PRS GPGS PAQWLLY THP THSWQRADLCAYLNPRP
PAPEA.
W SE PVLLAKGSAAYSDLQSMGIGPDGS PL FGCLYE.ANDYEE IVFLMFTLKQ.AFPA.EYLPQEPKS
SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYAS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
VYT L PP SREEMTKNQVS L T CLVKGFYP S D IAVEWE SNGQPENNYKT T P PVLDSDGS FFL
TSKLT
VDKSRWQQGNVFS CSVMHEALHNHYT QKS L S LS PGK
[0690] SEQ ID NO: 212:
g a t GCATC T C T GCCTTACC T GCAGAAAGAAAGCGT GIT CCAGT C TGGCGCCCACGCC
T.ACAG.AA
T T CCCGCT C T GC T GTAT C T GCCAGGCCAGCAGT C T C TGC T GGC T T TCGC T
GAACAGCGGGCCAG
CAAGAAGGAT GAGCAC GC C GAAC T GAT C G T GC T GC GGAGAGGC GAT TAC GAC GC C g g
cACACAT
CAGGT GCAGT GGCAGGC T CAAGAGGT GGT GGC T CAGGCTAGAC I GGAC GGCCACAGAT C TAT GA
ACCCCIGICCTCTGTA.CGA.TgaaCA.GA.CCGGCA.CA.CTGITTCTGITCTTTATCGCTATCCCCGG
C CAAGTGACCGAGCAGCAGCAGCT GCAGACAAGAGCCAACGT GACCAGAC T GIGT t a cGT GAC C
TCCACCGACCACGGCAGAACCIGGICTAGCCCTAGAGATCTGACCGACGCCGCCATCGGACCTG
CC TATAGAGAGT GGTCCACC T TCGCCGT T GGACC T GGACACIGT CTCCAGC T GCACGACAGGGC
TA.GAT CIC T GGT GGIGCCI GCCIA.CGCCI.AT.AGAAA.GCT GCACCCCAAA.CAGCGGCC TAT TCC
T
AGCGCC TIC T GC T TIC I GAGCCACGAT CACGGCAGGACAT GGGCCAGAGGACAT T T CGT GGCCC
AGGACACACTGGAATGCCAGGTGGCCGAAGTGGAAACCGGCGAGCAGAGAGTCGTGACCCTGAA
C GCC.AG.AT C T CAC C T GA.GA.T T CAGA.G GCA.GGCCC.AGA.GCACAAACGAC GGC C G GAT
T T C CAA.
GAGAGCCAGC T GGTCAAGAAACT GGT GGAACC T CC T CCAa ccGGCTGT CAGGGAAGCGT GAT CA
GC T T T CCAT C T CC TAGAAGC GGCCC T GGC IC TCC T GC T CAGT GGCTGC T
GTATACACAC CCCAC
ACACAGCT GGCAGAGAGCCGATCT GGGCGCC TACC I GAT CC T.AGACC T CC T GCT COT GAGGC T
T GGAGCGAACC T GTICIGC T GGCC.AAGGGCAGCgc t GCC TACAGCGAT C T GC.AGIC TAT
GGGCA.
CAGGCCCT GAT GGCAGCCC T C IGT IT GGC I GICT GIACGAGGCCAACGAC TACGAAGAGATCGT
GT T CC TGAT GT TCACCCTGAAGCAGGCCT T T CCAGCCGAGTACC TGCC T CAA.GAGCCCAAAT C T
T cTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCT
T CC T C T TCCCCCCAAAACCCAAGGACACCC T CAT GATCT CCCGGACCCC T GAGGT CACAT GCGT
GGT GGTGGAC GT GAGCCACGAAGACCC T GAGGT CAAGT T CAAC T GGTAC GT GGACGGCGT GGAG
G T GCAT.AA.T G C CAAGA.CAAAGCC GC GGGAGGA.GCA.G TA.0 g c cAGCA.0 G TAC C GI G
T GG T C.AGC G
TCCTCACCG,TCCTGCACCAGGACTGGCTC_IAATGGCAAGGAGTACAAC_:',TGCAAGGTCTCCAACAA
AGCCC TCCCAGCCCCCAT CGAGAAAAC CAT C T CCAAAGCCAAAGGGCAGCCCCGAGAAC CACAG
GT c TACACCC T GCCCCCAT CCCGGGAGGAGAT GACCAAGAACCAGGT CAGCC TGACC T GCCTGG
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TCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAA
C TACAAGACCACGCCTCCCGT GCT GGAC TCCGACGGC TCC T TC T TCC T C a ccAGCAAGC TCACC
GT GGACAAGAGCAGGIGGCAGCAGGGGAACGTC T TC TCAT GC TCCGT GAT GCAT GAGGC TCT GC
A_CAAC CAC TA_CAC GCAGAAG_AGC C IC TCCCT GT CTCC GGG TAAA_
106911 SEQ ID NO: 213:
EVQLVQS GAEVKKPGATVK I S CKVS G FN I KDTYMHWVQQAPGKGLEWMGL I DPANDNT I YAEKF
QGRVT I TADT S TDTAYMELS S LRSEDTAVYYCAREGYGGS YGEGYWGQGTLVTVS SAS TKGPSV
FPLAPS SKS T S GGTAALGCLVKDYFPE PVTVSWNS GALT SGVHT FPAVL QS S GLYS L S SVVTVP
S S S LGTQTY I CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVELFPPKPKDTLMI
S RT PE VICVVVDVSHE DPEVK FNWYVDGVEVHNAKTKPREE QYAS TYRVVSVLTVLHQDWLNGK
EYKCKVSNKAL PAR IEKT I SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLTSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GK
106921 SEQ ID NO: 214:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDE QT GIL FL FFIAI PGQVTEQQQLQTRANVTRLCYVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LN.ARS HLR FRVQAQS TNDGLDFQ
ESOLVKKT,VEPPPTGCOGSVISFPSPRSC_IPGSPAOWT,LYTHPTHSWORADLGAYLNPRPPAPF.A
W SE PVLLAKGSAAYSDLQSMGTGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQEPKS
SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNA.KTKPREEQY.ASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA.P I EKT I S KAKGQ PRE PQ
VYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLT
VDKSRWQQGNVFS CSVMHEALHNHYTQKS L S LS PGK
106931 SEQ ID NO: 215:
GAGGTGCAGCTGGITCAGTCTGGCGCCGAAGTGAAAAAGCCTGGCGCCACCGTGAAGATC.AGCT
GCAAGGTGT C C GGCT T CAACATCAAGGACACC TACATGCAC T GGGTGCAGCAGGCCCC T GGCAA
AGGAC T TGAAT GGATGGGCC T GATCGACCCCGCCAAC GACAATACCAT C TAC GCCGAGAAGT IC
CAGGCCAGAG T GACCAT CACCGCCGACACCICTACCGA.CACCGCCTACAT GGAAC T GAG CAGCC
T GAGAAGCGAGGACACCGCC G TGTAC TAC T GT GCCAGAGAAGGC TACGGCGGCAGC TAC GGCGA
AGGATAT T GGGGACAGGGCACCCIGGICACCGT TAGC TC T GCt a gcACCAAGGGCCCAT CcGTC
T TCCCCCIGGCACCCTCCTCCAAG.AGCACCICTGGGGGCACAGCGGCCCTGGGCTGCCTGGICA.
AGGACT.ACTTCCCCGAACCGGTGA.CGGTGICcTGG.AACTCAGGCGCtCTGACCA.GCGGCGTGCA
CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
TCCAGCAGCT T GGGCACCCAGACC TACATC T GCAACGT GAAT CACAAGCCCAGCAACAC CAAGG
T GGACAAGAAAGT TGAGCCCAAATCT T GT GACAAAAC TCACAC.ATGCCCACCGT GCCCAGCAC C
TGAACTCCTGGGGGGACCGTCAGICTICCICTICCCCCCAAAACCCAAGGACACCCTCATGATC
TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
T CAAC TGGTACGT GGACGGCC TGGA.GGT GCATAAT GC CAA.GACAAAGC C GC GGGAGGAG CAG
TA.
C gccAGCACGTACCGT GT GGTCAGCGTCC TCACCGTCCT GCACCAGGA_C T GGCT GAAT GGCAAG
GAGTACAAGTGCAAGGICTCCAAC.AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAG
C CAAAGGGCA_GCCCCGA GAA_C CACAGGT c T AC ACCC TGCCCCCATCCCGGGAGGA GAT GACCAA
GAACC.AGGTCAGCCTGA.CCTGCCIGGICAAAGGCTICTATCCC.AGCGACATCGCCGTGGAGIGG
GAGAGCAAT GGGCAGCCGGAGAACAAC TACAAGAC CACG CC T CC CGT GC T GGACT CCGACGGC T
CC T TC T TCC T C a ccAGCAAGC TCACCGT GGACAAGAGCAGGT GGCAGCAGGGGAACGT C T TC
TC
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AT GC T CCGT GAT GCAT GAGGC ICI GCACAACCAC TACAC GCAGAAGAGCC TC TCCC T GT C
TCCG
GGTAAA
106941 SEQ ID NO: 216:
g a t GCATC TC T GCCT TACC T GCAGAAAGAAAGCGT GT TCCAGTC TGGCGCCCACGCC TACAGAA
T TCCCGCTC T GC T GTATC T GCCAGGCCAGCAGTC TC TGC T GGC T T TCGC T
GAACAGCGGGCCAG
CAAGAA.GGAT GAGC.AC GC C GAAC T GAT CG T GC T GC GGA.GAGGC GAT TAC GAC GC C
ggcACACA.T
CAGGTGCA.GT GGC.AGGCTC.AAG.AGGTGGIGGCTCA.GGCTAGACIGGA.CGGCC.ACA.GAT C TAT CA
ACCCCTGICCTCTGTACGATgaaCAGACCGGCACACTGTTTCTGITCTTTATCGCTATCCCCGG
C CAAGTGACCGAGCAGCAGCAGCT GCAGACAAGAGCCAACGT GACCAGAC T GTGT t a cGT GAC C
TCCACCGACCACGGCAGAACCTGGTCTAGCCCTAGAGATCTGACCGACGCCGCCATCGGACCTG
CC TAT.AGAGAG T GGTCCACC T TCGCCGT T GG.ACC T GGACAC T GI CTCCAGC T
GCACGACAGGGC
TAGATC IC T GG T GGTGCC T GCCTACGCC TATAGAAAGCT GCACCCCAAACAGCGGCC TAT TCC T
AGCGCC TIC T GC T TIC T GAGCCACGATCACGGCAGGACAT GGGCCAGAGGACAT T TCGT GGCCC
AGGACA.CACTGGAATGCCAGGIGGCCGAAGIGGAAACCGGCGAGCAGAGAGTCGTGA.CCCTG.AA
C GC CAGAT C T CAC C T GA GAT TCAGAGT GCAGGCCCAGAGCACAAACGA_CGGCCT GGAT T T CC
AA
GAGAGCCAGCTGGICAAGAAACTGGTGGAACCTCCTCCAaccGGCTGTCAGGGAAGCGTGATCA.
GC T T TCCATC T CC TAG.AAGCGGCCCT GGC TC TCC T GC TCAGT GGCTGC T
GTATACACACCCCAC
ACACAGCT GGCAG.AGA.GCCGATCT GGGCGCC TA.CC T G.AATCC T.AGA.CC T CC T GCTCC T
GAGGC T
T GGAGCGAACC T GT= T GC T GGCCAAGGGCAGCgc t GCC TACAGCGAT C T GCAGTC TAT GGGCA
CAGGCCCT GAT GGCAGCCC T C TGT IT GGC T GTC T GTACGAGGCCAACGAC TACGAAGAGATCGT
GT TCC TGAT GT TCACCC T GAAGCAGGCC T T TCCAGCCGAGTACC TGCC T CAAGAGCCCAAATC T
T cTGACAAAACTOACACATGCCCACCGTGCCCAGCAOCTG.AACTCCTGGGGGGACCGTCAGICT
T CC TC T TCCCCCCAAAACCCAAGGACACCC TCAT GATCT CCCGGACCCC T GAGGTCACAT GCGT
GGTGGIGGACGTG.AGCCACGAAGA.CCCTGA.GGICAAGTTC.AACTGGTACGTCGA.CGGCGTGGAG
GTGCATAATGOOAAG'ACAAAGCCGCGGGAGGAGCAGTACgccAG'CACGTACCGTGTGG'TOAC'CG
T CC TCACCGT CC T GCAC CAG GACT GGC T GAAT GGCAAGGAG TACAAGT GCAAGGTC TCCAACAA
AGCCCTCCCAGCCCCCATCGAGAA.AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG
GT c TAC.ACCC T GCCCCCAT CCCGGGA.GGAGA.T GA.CC.AAGAACC.AGGTCAGCCIGt a c T
GCCTGG
T CAAAGGCT TO TAT =AGO. GACAT GO G T GGAG T GGGAGAGOAAT GG GCAGC C GGAGAACAA
CTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTICCTCTATAGC.AAGCTCACC
GT GGACAAGAGCAGGT GGCAGCAGGGGAACGT C T IC TCAT GOT CCGT GAT GCAT GAGGC T CT GC
ACAAC CAC TA_CAOGCAGAAGAGCC TAAGc T T GTC TCCGGGTAAA
106951 SEQ ID NO: 217:
DAS L PYLQDE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRASKKDEHAEL IVLRRGDYDAPTH
QVQWQA.QEVVAQ.ARLDGHRSMNPCPLYDEQT G TL FL FFIAI PCQVTEQQQLQTRANVTRLCQVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARS LVVPAYAYRKLHPKQRP P
SAFC FL SHDHGRTWARGH FVAQDT LANQVAEVE T GE QRVVT LNARS HLRARVQAQS TNDGLDFQ
ESQLVKKLVEPPPQGCQGSVI S FPS PRS GPGS PAQWLLY THP THSWQRADLCAYLNPRP PAPEA.
W SE PVLLA.KGSAAYSDLQSMGTGPDGS PL FGCLYE.ANDYEE IRFRMFT LKQ.AFPA.EYL PQEPKS
S DKTHTCPPC PAPELLGGPSVFL FPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPRE E QYAS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
VYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLT
VDKSRWQQGNVFS CSVMHEALHNHYTQKS L S LS PGK
106961 SEQ ID NO: 218:
g a t CCATC TC T GCCTTACC T GCAGga t GAAACCGT GITCCACTC TGGOGCCCACGCC TACAG.AA
T T CCCCCTC T GOT GTATC T GCCAGGCCAGCAGTC TC TGC T GGC T T TCGC T
GAACAGCGGGCCAG
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CAAGAAGGAT GAGCAC GC C GAAC T GAT CG T GC T GC GGAGAGGC GAT TAC GAC GC C c c
tACACAT
CAGGT GCAGT GGCAGGC T CAAGAGGT GGT GGC T CAGGCTAGAC T GGAC GGCCACAGAT C TAT GA
ACCCCTGICCTCTGTACGATgaaCAGACCGGCACACTGITTCTGITCTTTATCGCTATCCCCGG
CCAGTGACCGAGCAGCAGCAGCTGCAGACAPGAGCGAACGTGACCAGACTGTGTCAAGTGACC
T CCACCGACCACGGCAGAACC 'EGG= TAGCCC TAGAGAT C T GACCGACGCCGCCATCGGACC T G
CC TATAGAGAGT GGICCACC T TCGCCCTICCACCTCCACACTCTCTCCAGCTCCACGACAGGGC
T_AGATC TC T GGT GGTGCC T GCCTACGCC TATAGAAAGCT GCACCCCAAACAGCGGCC TAT TCC T
AGCGCC T TC T GC T T TC T GAGCCACGATCACGGCAGGACAT GGGCCAGAGGACAT T TCGT GGCCC
AGGACACACTGgcgaat CAGGTGGCCGAAGTGGAAACCGGCGAGCAGAGAGTCGTGACCCTGAA
C GC CAGAT C T CAC C T GAGAG C CAGAG T GCAGGCCCAGAGCACAAACGACGGCCT GGAT T
TCCAA
GAGAGCCAGC T GGTCAAGAAACT GGT GGAACC T CC T CCACAGGGCTGT CAGGGAAGCGT GAT CA
GC T T TCCATC T CC TAGAAGCGGCCCT GGC TC TCC T GC TCAGT GGCTGC T
GTATACACACCCCAC
ACACAGCT GGCAGAGAGCCGATCT GGGCGCC TACC T GAATCC TAGACC T CC T GCTCC T GAGGC T
T GGAGCGAACC T GITC T GC T GGCCAAGGGCAGCgc t GCC TACAGCGAT C T GCAGTC TAT
GGGCA
CAGGCCCT GAT GGCAGCCC T C TGT T T GGC T GTC T GTACGAGGCCAACGAC TACGAAGAGATCc g
t T TCc gtAT GT TCACCCTGAAGCAGGCCITTCCAGCCGAGTACCTCCCTCAAGAGCCCAAATCT
T cTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCT
T CC TC T TCCCCCCAAAACCCAAGGACACCC TCAT GATCT CCCGGACCCC T GAGGTCACAT GCGT
GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
G T GCATAAT GC CAAGACAAAGCCGCGGGAGGAGCAGTAC g c cAGCAC G TAC C GT GT GG T
CAGCG
T CC TCACCGT CC T GCAC CAG GACT GGC T GAAT GGCAAGGAG TACAAGT GCAAGGTC TCCAACAA
AGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG
GT c TACACCC T GCCCCCAT CCCGGGAGGAGATCACCAAGAACCACCTCAGCC TCt a c T GCCT GC
T CAAAGGCT T C TAT CC CAGC GACAT C GC C G T GGAG T GGGAGAGCAAT GG GCAGC C
GGA_GAACAA
CTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACC
GT GGACAAGAGCAGGT GGCAGCAGGGGAACGTC T TC TCAT GC TCCGT GAT GCAT GAGGC TCT GC
ACAAC CAC TACACGCAGAAGAGCC TAAGc T TGICICCGGGTAAA
[0697] SEQ ID NO: 219:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFAE QRRSKKDEHAEL IVLRRGDYDAGTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDEQT GIL FL FFIAI PGQVTEQQQLQTRANVTRLCYVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLR FRVQAQS TNDGLDFQ
E S QLVKKLVE P PP TGCQGSVI S FPS PRS GPGS PAQWLLY THP THSWQRADLGAYLNPRP PAPEA
W SE PVLLAKGSAAYSDLQSMGTGPDGS PL FGCLYEANDYEE IVFLMFTLKQAFPAEYLPQEPKS
SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I S KAKGQ PRE PQ
VYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLT
VDKSRWQQGNVFS CSVMHEALHNHYTQKS L S LS PGK
[0698] SEQ ID NO: 220:
g a t GCATC TC T GCCT TACC T GCAGAAAGAAAGCGT GT TCCAGTC TGGCGCCCACGCC TACAGAA
T TCCCGCTC T GC T GTATC T GCCAGGCCAGCAGTC TC TGC T GGC T TTCGC T
GAACAGCGGcggAG
CAAGAAGGAT GAGCAC GC C GAAC T GAT C G T GC T GC GGAGAGGC GAT TAC GAC GC C g g
cACACAT
CAGGT GCAGT GGCAGGC TCAAGAGGT GGT GGC TCAGGCTAGAC T GGACGGCCACAGAT C TAT GA
ACCCCTGICCTCTGTACGATgaaCAGACCGGCACACTGITTCTGTICTTTATCGCTATCCCCGG
C CAAG T GACC GAG CAG CAG CAGC T GCAGACAAGAGCCAAC G T GAC CAGAC T GIGT
tacGTGACC
TCCACCGACCACGGCAGAACCTGGTCTAC_1CCCTAGAGATCTGACCGACGCCGCCATCGGACCTG
CC TATAGAGAGT GGTCCACC T TCGCCGTTGGACCTGGACACTGTCTCCAGCTGCACGACAGGGC
TAGATC TC T GGT GCTGCC T GCCTACGCC TATAGAAACCT GCACCCCAAACAGCCGCC TAT TCC T
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AGCGCC T TC T GC T TIC T GAGCCACGAT CACGGCAGGACAT GGGCCAGAGGACAT T T CG T
GGCCC
AGGACACAC T GGAATGCCAGG TGGCCGAAGT GGAAACCGGCGAGCAGAGAGT CGT GACCC TGAA
C GC CAG.AT C T CAC C T GAGA.T T CAGA.GT GCA.G GC C C.AGA.G CACAAAC GAC G GC C
T G GAT T T C CAA
GA_GAGCCAGC T GGICAAGAAACT GGT GGAACC T CC T CCAa coGGCTGT CAGGGAAGCG T GAT
CA_
GC T T T CCAT C T CC TAG.AAGCGGCCCT GGC T C T CC T GC T CAGT GGCTGC T
GTATACACACCCCAC
ACACAGCT GGCAGAGAGCCGATCT GGGCGCC TACC T GAAT CC TAGACC T CC T GCT COT GAGGC T
T GGAGCGAACC T GT TC T GC T GGCCAAGGGCAGCgc t GCC TACAGCGAT C T GCAGTC TA_T
GGGCA
CAGGCCCT GAT GGCAGCCC T C TGT IT GGC T GTC T GTACGAGGCCAACGAC TACGAAGAGATCGT
G T T CC TGAT G T TCACCCTGAAGCAGGCCITTCCAGCCGAGTACCTGCCTCAA.GAGCCC.AAATCT
T cTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCT
T CC T C T TCCCC CCAAAACCCAAGGACACCC T CAT GATCT CCCGGACCCC T GAGGT CACAT GCGT
GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
G T GCA.T.AAT G C CAAGACAAAGCC GCGGGAGGA.GCA.G TA.0 g c cAGCA.0 G TAC C GT GT
GG T C.AGCG
T CC T CA CCGT CC T GCAC CAGGACT GGC T GAAT GGCAAGGAGTACAAGT GCAAGGTC T
CCAACAA
AGCCC TCCCAGCCCCCAT CGAGAAAAC CAT C T CCAAAGCCAAAGGGCAGCCCCGAGAAC CACAG
GT c TACACCC T GCCCCCAT CCCGGGAGGAGAT GACCAAGAACCAGGT CAGCCIGt a c T GCCIGG
T CAAAGGC T T C TATCCCAGC GACATCGCC GT GGAGT GGGAGAGCAAT GGGCAGCCGGAGAACAA
C TACAAGACCACGCCT CCCG T GCT GGAC T CCGACGGC T CC TTCT TCC T C TATAGCAA.GC T
CACC
G T GGACAAGAGCAGGT GGCAGCAGGGGAACGT CITC TCAT GOT CCGT GAT GOAT GAGGC T CT GC
AC.AA.CC.AC TACACGCA.GAA.GAGCC T.AA.Gc T T GT C T CC GG G TAAA.
[0699] SEQ ID NO: 221:
AS TKGP SVFP LAP S SKS TS GG TAALGCLVKDYFPE PVTVSWNS GALT S GVHT FPAVLQS SGLYS
LSSVVTVPS S S LGTQTY CNVNHKPSNTKVDKKVE PKS CDKTHT CPPC PAPELLGGP SVFL FP P
KPKDTLMI SRT PEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPRE E QYNS TYRVVSVLTVL
HQDWLNGKEYKCKVSNKAL P_AP EKT I SK_AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKT T PPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK
[0700] SEQ ID NO: 222:
A_S TKGP SVFP LAP S SKS TS GG TAALGCLVKDYFPE PVTVSWNS GALT S GVHT FPAVLQS
SGLYS
LSSVVTVPS S S LGTQTY I CNVNHKPSNTKVDKKVE PKS CDKTHT CPPC PAPELLGGP SVFL FP P
KPKDTLMI SRT PEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPRE E QYAS TYRVVSVLTVL
HQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKT T PPVLDSDGS FFLYSKL TVDKSRWQQGNVFS CSVMHEALHNHYT
QKSLSLSPGK
107011 SEQ ID NO: 223:
AS TKGP SVFP LAP S SKS TS GG TAALGCLVKDYFPE PVTVSWNS GALT S GVHT FPAVLQS SGLYS
LSSVVTVPS S S LGTQTY I CNVNHKPSNTKVDKKVE PKS CDKTHT CPPC PAPELLGGP SVFL FP P
KPKDTLMI SRT PEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPRE E QYNS TYRVVSVLTVL
HQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
P S D TAVEWE SNGQPENNYKT T PPVLDSDGS FFLTSKLTVDKSRWQQGNVFSCSVMHEALFINTHYT
QKSLSLSPGK
107021 SEQ ID NO: 224:
AS TKGP SVFP LAP S SKS TS GGTAALGCLVKDYFPEPVTVSWNSG.ALTS GVHT FPAVLQS SGLYS
LSSVVTVPS S S LGTQTY CNVNHKPSNTKVDKKVE PKS CDKTHT CPPC PAPELLGGP SVFL FP P
186
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KPKDTLMI SRT PEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPRE E QYNS TYRVVSVLTVL
HQDWLNGKEYKCKVSNKAL PAP I EKT I S KAKGQPRE PQVY T L PP SREEMTKNQVS LYCLVKGFY
PSDIAVEWESNGQPENNYKT T PPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALLINHYT
QKSLSLSPGK
107031 SEQ ID NO: 225:
AS TKGP SVFP LAP S SKS TS GGTAALGCLVKDYFPE PVTVSWNS GALT S GVHT FPAVLQS SGLYS
LS SVVTVPS S S LGTQTY I CNVNHKPSNTKVDKKVE PKS CDKTHT CPPC PAPELLGGP SVFL FPP
KPKDTLMI SRT PEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPRE E QYAS TYRVVSVLTVL
HQDWLNGKEYKCKVSNKAL PAP I EKT I S KAKGQPRE PQVY T L PP SREEMTKNQVS L T CLVKGFY
PSDIAVEWESNGQPENNYKT T PPVLDSDGS PFLTSKLTVDKSRWQQGNVPSCSVMHEALHNHYT
QKSLSLSPGK
107041 SEQ ID NO: 226:
AS TKGP SVFP LAP S SKS TS GGTAALGCLVKDYFPE PVTVSWNS GALT S GVHT FPAVLQS SGLYS
LS SVVTVPS S S LGTQTY I CNVNHKPSNTKVDKKVE PKS CDKTHT CPPC PAPELLGGP SVFL FPP
KPKDTLMI SRT PEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPRE E QYAS TYRVVSVLTVL
HQDWLNGKEYKCKVSNKAL PAP I EKT I S KAKGQPRE PQVY T L PP SREEMTKNQVS LYCLVKGFY
PSDIAVEWESNGQPENNYKT T PPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK
107051 SEQ ID NO: 227:
AS TKGP SVFP LAP S SKS TS GGTAALGCLVKDYFPE PVTVSWNS GALT S GVHT FPAVLQS SGLYS
LS SVVTVPS S S LGTQTY I CNVNHKPSNTKVDKKVE PKS CDKTHT CPPC PAPELLGGP SVFL FPP
KPKDTLMI SRT PEVTGVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKAL PAP I EKT I SKAKGQPRE PQVY T L PP SRDE L TKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKT T PPVLDSDGS FFLYSKL TVDKSRWQQGNVFS CS VMHEALHNHYT
QKSLSLSPGK
107061 SEQ ID NO: 228:
AS TKGPSVFPLAPCSRS TSES TAALGCLVKDYPPE PVTVSWNS GALT S GVIIT FPAVLQS SGLYS
LS SVVTVPS SNFGTQTYT CNVDHKPSNTKVDKTVERKCCVECPPCPAP PVAGPSVFL FP PKPKD
T LM I SRTPEVT CVVVDVS HE DPEVQFNWYVDGVEVHNAKTKPREEQFNS T FRVVSVL TVVHQDW
LNGKEYKCKVSNKGLPAP I EKT I SKTKGQPRE PQVYTLP P SREEMTKNQVS L TCLVKG FYPS D I
SVEWE SNGQPENNYKT TPPMLDS DGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
S LS PGK
107071 SEQ ID NO: 229:
AS TKGPSVFPLAPCSRS TS GGTAALGCLVKDYFPE PVTVSWNS GALT S GVHT FPAVLQS SGLYS
LS SVVTVPS S S LGTQTYTCNVNHKPSNTKVDKRVELKTPLGDT THTCPRCPE PKS CDT PPPCPR
C PE PKSCDT P P PCPRCPE PKS CDT PPPCPRCPAPELLGGP SVFL FPPKPKDT LMI SRT PEVT CV
VVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNS TFRVVSVLTVLHQDWLNGKEYKCKVSNK
AL PAP IEKT I SKTKGQPRE PQVYTLPPSREEMTKNQVSL TCLVKGFYPS D IAVEWES S GQPENN
YNT TPPMLDS DGS FFLYSKL TVDKSRWQQGNI FS CSVMHEALHNRFTQKS L S LS PGK
187
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107081 SEQ ID NO: 230:
AS TKGPSVFPLAPGSRS TSES TAALGCLVKDYFPE PVTVSWNS GALT S GVHT FPAVLQS SGLYS
LS SVVTVPS S S LGTKTYT CNVDHKPSNTKVDKRVE SKYGP PCP S CPAPE FLGGPSVFL FP PKPK
D T LM I SRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRVVSVL TVLHQD
WLNGKEYKGKVSNKGL PS S I EKT I SKAKGQPRE PQVYT L P PS QEEMTKNQVS LT CLVKG FYP S
D
IAVEWESNGQPENNYKT TPPVLDSDGS FFLYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKS
LSLSLGK
107091 SEQ ID NO: 231:
TVAAPSVFI FP P S DEQLKS G TASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDS T
YSLS S TLTLSKADYEKHKVYACEVTHQGLS SPVTKS FNRGEC
107101 SEQ ID NO: 232:
RTVAAP SVF I FP P SDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDS
TYSLS STLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC
107111 SEQ NO: 233:
GQPKANPTVTL FP PS SEELQANKATLVCL I SDFYPGAVTVAWKADGSPVKAGVET TKPSKQSNN
KYAAS SYLS L T PEQWKSHRSYSCQVTHEGS TVEKTVAPT EC
107121 SEQ ID NO: 234:
DAS L PYLQKE SVFQSGAHAYR I PALLYL P GQQS LLAFA_E QRRSKKDEHAEL IVLRRGDYDACTH
QVQWQAQEVVAQARLDGHRSMNPCPLYDEQT GT L FL FFIAI PGQVTEQQQLQTRANVTRLCYVT
S TDHGRTWS S PRDLTDAAI GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP I P
SAFC FL SHDHGRTWARGH FVAQDT LE CQVAEVE T GE QRVVT LNARS HLR FRVQAQS TNDGLDFQ
E SQLVEKLVE PPPTGCQGSVI S FP S PRS GPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA
W SE PVLLAKGSAAYSDLQSMG TGPDGS PL FGCLYEANDYEE IVELMFTLKQAFPAEYLPQ
107131 SEQ ID NO: 235:
EVQLVQS GAEVKKPGATVK I S CKVS G FN KDTYMHWVQQA PGKGLEWMGL I DPANDNT I YAEKF
QGRVT I TADT S TDTAYMELS SLRSEDTAVYYCAREGYGGSYGEGYWGQGTLVTVS SAS TKGPSV
FPLA P S SKS T S GGTAALGCLVKDYFPE PVTVSWNS GALT SGVHT FPAVL QS SGLYSLS SVVTVP
S S S LGTQTY I CNVNHKP SNTKVDKKVE PKS CDKTHT CPP C PAPELLGGP SVFL FP PKPKDTLMI
S RT PE VTCVVVDVSHE DPEVK ENWYVDGVEVHNAKTKPREE QYNS TYRVVSVLTVLHQDWLNGK
E YKCKVSNKAL RAP I EKT I SKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPS D IAVEW
SNGQPENNYKT TPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSP
GK
107141 SEQ ID NO: 236:
EVQLX1X2SGAEX3X4KPGAX5VX6X7SCX8X9SGFNIKDTYMHWVX10QXl_PX12X13GLEWX14GX15
I DPANDNIX16YX17X16KFQX19X2oX21T I TAD15X22D1AYX23X2 iLSSLX25SEDTAVYYCAREGY
GGS YGEGYWGQGTX26X27 TVS S
188
CA 03173557 2022- 9- 27
WO 2022/150521
PCT/US2022/011504
107151 SEQ ID NO: 237:
Xi IVMTQX2PX3X4LX5X6SX7GX8RVTX9X iDCX1LASQSVSNDX12X13WYQQKPGQX14 PX15LL I YY
AS IRFTGX16PX17RFX18GS GX19G1DF1X20T IX01X22X23QX24EDX05AVYX060QQDYX07SPWT F
GX26GTKx29EIK
107161 SEQ ID NO: 238:
X iX2SX3X4X5LQX6ESVFQS GAHAYRI PAL LYL PGQQSLLAFAEQRX7 SX8X9DEHAEL IVX loRRG
DYDAX THQVQWX12AQEVVAQAX 13LX14GHRSMNPCPLYDX15QT GTL FL FFIAI PX16X17VTEX
18QQLQTRANVIRLX29X20VT S TDHGRTWSSPRDLTDAAIGPX22YREWST FAVGPGHX22LQLHDX
23X24RSLVVPAYAYRKLHPX25X26X27P I P SAFX2 a FL SHDHCRTWARCH FVX29QDTX3 oECQVAEV
X31TGEQRVV1LNARSX32X33X34X35RX36QAQSX37NX38GLDFQX39X40QX42VKKLX42EPPPX43G
X44QGSVI S FPS PRS GPGSPAQX45LLYTHPTHX46X47QRADLGAYLNPRPPAPEAWSEPX48LLAK
GSX49AYS DLQSMGT GPDGS PL FGX5oLYEANDYEE I X5iFX52MFTLKQAFPAEYL PQ
107171 SEQ ID NO: 239:
X lASLPX2LQX3E SVFQS GAHAYRI PALLYLPGQQSLLAFAEQRX4SKKDEHAEL IVLRRGDYDAX
5 THQVQWQAQEVVAQARL DGHRSMNPCPLYDX 6QT GIL FL FFIAI PGQVTEQQQLQTRANVTRLC
X7VTSTDHGRTWSSPRDL TDAA GPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPX8Q
RP I PSAFC FL SHDHGRTWARGHFVAQDTLECQVAEVET GEQRVVTLNARSHLRX9RVQ.AQS TNDG
LDFQESQLVKKLVEPPPX10GCQGSVI S FPS PRS GPGS PAQWL LYTHP THX 11X19QRADL GAYLN
PRPPAPEAWSEPVLLAKGSX23AYSDLQSMGTGPDGSPLFGCLYEANDYEE I X 14 FX25MFTLKQA.
FPAEYLPQ
107181 SEQ ID NO: 240:
X iX2SX3X4X5LQX6ESVFQS GAHAYRI PAL LYL PGQQSLLA FAEQRX7SX8X9DEHAEL IVX1 oRRG
DYDAX 12 THQVQWX22AQEVVAQAX23LX24GHRSMNPCPLYDX25QT GTL EL FFIAI PX26)(17VTEX
16QQLQTR7\NVIRLX19X20VT S TDHGRTWS S PRDL TULA.' GPX21YREWST FAVGPGHX22LQLHDX
23X24RSLVVPAYAYRKLHPX25X26X27P I P SAFX28 FL SHDHGRTWARGH FVX29QDTX30ECQVAEV
X311GEQRVVTLNAR5X32X33X34X35RX36QAQ5X37NX38GLDFQX39X40QX41VKKLX42EPPPX43G
X44QGSVI S FPS PRS GPGSPAQX45LLYTHPTHX46X47QRADLGAYLNPRPPAPEAWSEPX48LLAK
GSX49AYS DLQ5'MGT GPDGS PL FGX5uLYEANDYEE I X5iFX52MFTLEQAFPAEYL PQX53DKTIIT C
PPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAP IEKT I SKAKGQPRE PQVY TLPPS
REEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKT T PPVLDSDGS FEL TSKL TVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK
107191 SEQ ID NO: 241:
X lASLPX2LQX3E SVFQS GAHAYRI PALLYLPGQQSLLAFAEQRX4SKKDEHAEL IVLRRGDYDAX
5 THQVQWQAQEVVAQARL DGHRSMNPCPLYDX 6QT GIL FL FFIAI PGQVTEQQQLQTRANVTRLC
X7VISTDHGRTWSSPRDL TDAAIGPAYREWS T FAVGPGHCLQLHDRARSLVVPAYAYRKLHPX8Q
RP I PSAFC FL SHDHGRTWARGHFVAQDTLECQVAEVET GEQRVVTLNARSHLRX9RVQAQS TNDG
LDFQESQLVKKLVEPPPX20GCQGSVI S FPS PRS GPGS PAQWL LYTHP THX12X12QRADL GAYLN
PRPPAPEAWS E PVLLAKGS Xi3AYSDLQSMGT GPDGS PL FGCLYEANDYEE X 14 FX15MFTLKQA
FPAEYLPQKioDKTHTCPPCPAPELLGGPSVFLFPPKPKDTIMI SRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKT I SK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGPYPSDIAVEWESNGQPENNYKTTPPVLDSDG
S FEL T SKL TVDKSRWQQGNVESCSVMHEALHNHYTQKS L SLS PGK
189
CA 03173557 2022- 9- 27
WO 2022/150521
PCT/US2022/011504
[0720] SEQ ID NO: 242:
EIVMTQSPPTLSLSPGERVTLSCRASQSVSNDLSWYQQKPGQAPRLLIYYASTRATGIPARFSG
SGSGTLFTLTISSLQPEDFAVYYCQQDYNSPWTFGQGTKVEIK
[0721] SEQ ID NO: 243:
EIVMTQSPPTLSLSPGERVTLSCRASQSVSNDLSWYQQKPGQAPRLLIYYASIRATGIPARFSG
SGSGTDFTLTISSLQPEDFAVYYCQQDYNSPWTFGQGTKVEIK
[0722] SEQ ID NO: 244:
EIVMTQSPPTLSLSPGERVTLSCRASQSVSNDLSWYQQKPGQAPRLLIYYASTRFTGIPARFSG
SGSGTDFTLTISSLQPEDFAVYYCQQDYNSPWTFGQGTKVEIK
[0723] SEQ ID NO: 245:
EIVMTQSPPTLSLSPGERVTLSCRASQSVSNDLSWYQQKPGQAPRLLIYYASTRATGIPARFSG
SGYGTDFTLTISSLQPEDFAVYYCQQDYNSPWTFGQGTKVEIK
[0724] SEQ ID NO: 246:
EIVMTQSPPTLSLSPGERVTLSCRASQSVSNDLSWYQQKPGQAPRLLIYYASTRATGIPARFSG
SGSGTDFTLTISSLQPEDFAVYFCQQDYNSPWTFGQGTKVEIK
[0725] SEQ ID NO: 247:
EIVMTQSPPTLSLSPGERVTLSCRASQSVSNDLSWYQQKPGQAPRLLIYYASIRATGIPARFSG
SGYGTDFTLTISSLQPEDFAVYYCQQDYNSPWTFGQGTKVEIK
[0726] SEQ ID NO: 248:
EIVMTQSPPTLSLSPGERVTLSCRASQSVSNDLSWYQQKPGQAPRLLIYYASIRATGIPARFSG
SGSGTDFTLTISSLQPEDFAVYFCQQDYNSPWTFGQGTKVEIK
[0727] SEQ ID NO: 249:
EIVMTQSPPTLSLSPGERVTLSCRASQSVSNDESWYQQKPGQAPRLLIYYASIRATGIPARFSG
SGYGTDFTLTISSLQPEDFAVYFCQQDYNSPWTFGQGTKVEIK
[0728] SEQ ID NO: 250:
DTYMH
[0729] SEQ ID NO: 251:
RIDPANDNTKYDPKFQD
107301 SEQ NO: 252:
L I DPANDNT I YAEKFQG
190
CA 03173557 2022- 9- 27
WO 2022/150521
PCT/US2022/011504
[0731] SEQ ID NO: 253:
KASQSVSNDVI
[0732] SEQ ID NO: 254:
YAS IRFT
[0733] SEQ ID NO: 255:
RAS QSVSNDL S
191
CA 03173557 2022- 9- 27
